Human Vocal Fold Research Articles

An acoustic pressure pipette aspiration method for isotropic materials

A measurement setup is presented as a method to determine the elasticity modulus of both artificial and real tissue as a function of frequency. The future goal at the end is to develop and validate the method to measure the elasticity modulus of ex vivo cadaver animal vocal folds over the human phonation frequency range. In the present study, we investigate an innovative acoustic pressure pipette aspiration technique to measure the material characteristic of an isotropic silicone specimen with similar characteristics as human vocal folds. With the aim of ensuring reliable results, we test our method on different mixtures of silicone rubber. Four silicone samples with different consistencies were tested over a frequency range of 50 –500Hz. Influence of vibrometer laser spot location in an enclosed area on the sample surface was investigated by repeating the measurements ten times. The results show only small standard deviations demonstrating the potential of the acoustic pressure pipette aspiration technique for material characterization.

Implementing Efficient Peptoid‐Mediated Delivery of RNA‐Based Therapeutics to the Vocal Folds

ObjectiveWe hypothesize that Smad3 is a master regulator of fibrosis in the vocal folds (VFs) and RNA‐based therapeutics targeting Smad3 hold therapeutic promise. Delivery remains challenging. We previously described a novel synthetic peptoid oligomer, lipitoid L0, complexed with siRNA to improve stability and cellular uptake. An advantage of these peptoids, however, is tremendous structural and chemical malleability to optimize transfection efficiency. Modifications of L0 were assayed to optimize siRNA‐mediated alteration of gene expression.MethodsIn vitro, Smad3 knockdown by various lipitoid variants was evaluated via quantitative real‐time polymerase chain reaction in human VF fibroblasts. Cytotoxicity was quantified via colorimetric assays. In vivo, a rabbit model of VF injury was employed to evaluate the temporal dynamics of Smad3 knockdown following injection of the L0‐siRNA complex.ResultsIn vitro, similar reductions in Smad3 expression were established by all lipitoid variants, with one exception. Sequence variants also exhibited similar nontoxic characteristics; no statistically significant differences in cell proliferation were observed. In vivo, Smad3 expression was significantly reduced in injured VFs following injection of L0‐complexed Smad3 siRNA at 1 day postinjection. Qualitative suppression of Smad3 expression persisted to 3 days following injury, but did not achieve statistical significance.ConclusionsIn spite of the chemical diversity of these peptoid transfection reagents, the sequence variants generally provided consistently efficient reductions in Smad3 expression. L0 yielded effective, yet temporally limited knockdown of Smad3 in vivo. Peptoids may provide a versatile platform for the discovery of siRNA delivery vehicles optimized for clinical application.Level of EvidenceNA

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

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

Acoustic Pressure Pipette Aspiration Method Combined with Finite Element Analysis for Isotropic Materials

A measurement setup combined with a numerical simulation by a linear finite element analysis is presented as a method to determine the elastic modulus of both artificial and real tissue as a function of frequency. At the end, the future goal is to develop and validate the method to measure the elastic modulus of in-vivo human vocal folds over the human phonation frequency range. In the present study, a miniaturized acoustic pressure pipette aspiration technique is developed to measure the material characteristics of an isotropic silicone specimen with similar characteristics as human vocal folds. In previous studies, friction and compression force effects of the pipette tip wall on the surface of the sample and the radius of the pipette were not investigated. Moreover, the large scale of the measurement setups made them impossible to use for clinical applications. Therefore, two different pipette sample cross-section boundary conditions and two different pipette radii were used. With the aim of ensuring reliable results, we tested our method with pipettes of two different radii on four silicone samples with different consistencies over a frequency range of 50–500 Hz. The simulation verified the measurement results in which the strong dependency of the elastic modulus on the excitation frequency, radius of the pipette, the pipette tip compression force and friction was revealed. By the simulation results, two different frequency dependent equations were developed for calculating elastic modulus of the silicone mixtures in the two cross-section boundary conditions. It was concluded that using a very small gap in between the pipette tip and the specimen can cancel the impact of the pipette tip force and friction which are the major cause of uncertainty. However, if a connection between the pipette and the surface is unpreventable, the contact force should be restricted to be absolutely zero.

Magnetic resonance imaging of the vocal fold oscillations with sub-millisecond temporal resolution.

The temporal resolution of the MRI acquisition is intrinsically limited by the duration of the spatial encoding, which is typically on the order of milliseconds. Faster motion such as the vibration of the vocal folds during phonation cannot be imaged with conventional MRI as this would require sampling frequencies in the kilo-Hertz range. Here, a faster MRI acquisition strategy is presented that encodes a 1D periodic motion at a temporal resolution that is an order of magnitude higher compared to conventional MRI. The proposed method encodes the position of an object moving along 1 dimension by applying very short phase encoding gradients along the same direction. This reduces the temporal resolution from the repetition time (TR) to the duration of the phase encoding gradients, which in this work was well below 1 ms. The technique is applied to the vocal fold oscillations and the position of the vocal folds is measured simultaneously using electroglottography (EGG). Simulations of the point spread function for regular encoding and the proposed method are performed as well. With this new phase, encoding strategy oscillations of the human vocal folds up to a frequency of 145 Hz could be dynamically imaged at 10 images per cycle. Simulations show the advantage of this method over conventional imaging of fast moving objects. A new method for MR imaging of fast moving spins is presented allowing a temporal resolution below 1 ms at a spatial resolution below 1 mm, circumventing TR as the limit for temporal resolution.

Scattering properties and femtosecond laser ablation thresholds of human and canine vocal folds at 776-nm wavelength

.Ultrafast laser ablation may provide a treatment for vocal fold (VF) scarring. Optical properties of VFs must be known prior to clinical implementation to select appropriate laser surgery conditions. We present scattering lengths of epithelium , superficial lamina propria , and ablation thresholds of human and canine VF tissues. Our experimental approach involves an image-guided, laser-ablation-based method that allows for simultaneous determination of and in these multilayered tissues. Studying eight canine samples, we found , , , and . Studying five human samples, we found and . We studied the effects of cumulative pulse overlap on ablation threshold and found no significant variations beyond 12 overlapping pulses. Interestingly, our studies about the effect of sample storage on the scattering properties of porcine VF show a 60% increase in for fresh porcine VF when compared to the same sample stored in isotonic solution. These results provide guidelines for clinical implementation by enabling selection of optimal laser surgery parameters for subsurface ablation of VF tissues.

Induction of Fibrogenic Phenotype in Human Mesenchymal Stem Cells by Connective Tissue Growth Factor in a Hydrogel Model of Soft Connective Tissue.

Scar formation is the typical endpoint of wound healing in adult mammalian tissues. An overactive or prolonged fibrogenic response following injury leads to excessive deposition of fibrotic proteins that promote tissue contraction and scar formation. Although well-defined in the dermal tissue, the progression of fibrosis is less explored in other connective tissues, such as the vocal fold. To establish a physiologically relevant 3D model of loose connective tissue fibrosis, we have developed a synthetic extracellular matrix using hyaluronic acid (HA) and peptidic building blocks carrying complementary functional groups. The resultant network was cell adhesive and protease degradable, exhibiting viscoelastic properties similar to the human vocal fold. Human mesenchymal stem cells (hMSCs) were encapsulated in the HA matrix as single cells or multicellular aggregates and cultured in pro-fibrotic media containing connective tissue growth factor (CTGF) for up to 21 days. hMSCs treated with CTGF-supplemented media exhibited an increased expression of fibrogenic markers and ECM proteins associated with scarring. Incorporation of α-smooth muscle actin into F-actin stress fibers was also observed. Furthermore, CTGF treatment increased the migratory capacity of hMSCs as compared to the CTGF-free control groups, indicative of the development of a myofibroblast phenotype. Addition of an inhibitor of the mitogen-activated protein kinase (MAPK) pathway attenuated cellular expression of fibrotic markers and related ECM proteins. Overall, this study demonstrates that CTGF promotes the development of a fibrogenic phenotype in hMSCs encapsulated within an HA matrix and that the MAPK pathway is a potential target for future therapeutic endeavors towards limiting scar formation in loose connective tissues.

Metabolic activity of cells in the macula flava of the human vocal fold from the aspect of mitochondrial microstructure.

ObjectivesThere is growing evidence to suggest that the cells in the maculae flavae of the vocal fold mucosa are tissue stem cells of the human vocal fold. This study investigated the metabolic activity of the cells in the maculae flavae of the human vocal fold from the aspect of mitochondrial microstructure.Study DesignHistologic analysis of the human vocal folds.MethodsFive normal human adult vocal folds obtained from autopsy cases were investigated under transmission electron microscopy.ResultsMitochondria were randomly distributed in the cytoplasm of the cells. The morphological features of the mitochondria consisted of a double‐membrane‐bounded body containing matrices and a system of cristae. In each mitochondria, the lamellar cristae were sparse. The intercristal space was occupied by a mitochondrial matrix which contained electron‐dense matrix granules, mitochondrial DNA, and ribonucleoprotein granules. Some mitochondria spread out over or fused to the surface of a lipid droplet in the cytoplasm. In addition, both the mitochondrial outer and inner membranes and the membranes of the lipid droplets had disappeared. Some close association between mitochondria and rough endoplasmic reticulum was present.ConclusionsThe features of the mitochondria of the cells in the maculae flavae of the human vocal fold suggested that their metabolic activity and oxidative phosphorylation were low and that they may have shifted to the utilization of lipids to some extent for their metabolic needs.Level of EvidenceNA

Structural constitutive modeling of the anisotropic mechanical properties of human vocal fold lamina propria.

The anisotropic mechanical properties of the vocal fold lamina propria play an important role in voice production and control. The goal of this study is to develop a constitutive model capable of predicting lamina propria elastic moduli along both the longitudinal and transverse directions under different conditions of vocal fold elongation, which can be used as input to reduced-order phonation models based on linear elasticity. A structurally-based constitutive model that links microstructural characteristics of the lamina propria to its macromechanical properties is proposed. The model prediction has been shown to agree reasonably well with recent biaxial tensile testing results.

Magnetic Tissue Engineering of the Vocal Fold Using Superparamagnetic Iron Oxide Nanoparticles.

Losing one's ability to speak, because of tissue deficiency at the vocal fold (VF), leads to serious impairment in the quality of life. Until now, there is no successful approach for regenerating the VF. The aim of this study was to show the advantage of magnetic nanoparticles in the generation of scaffold-free three-dimensional (3D) VF cell constructs by magnetic tissue engineering (MTE). Rabbit VF fibroblasts were used to establish MTE: after cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs), cells can be controlled with a magnetic field thereby forming solid 3D cell structures. To transfer this method into human cells, SPIONs were adapted accordingly and tested for their influence on human VF (hVF) cells and for their ability to perform MTE with hVF cells. Of interest, the cell number and the magnet's shape influence the form of the rabbit VF cell construct. After successful characterization of hVF cells, biocompatibility analyses revealed no significant influence of SPIONs on them, thus 3D hVF cell constructs could be successfully generated by MTE. These basic results are important to develop MTE as an innovative method to regenerate functional VFs. We expect that in vivo studies, including MTE as an elegant, far-field controlled and touchless technology, will translate MTE VF bioconstructs into reconstructive laryngeal medicine. Impact Statement This study aims at nanotechnology for regenerative medicine by magnetic tissue engineering (MTE). New approaches for vocal fold (VF) reconstruction are desperately needed. Superparamagnetic iron oxide nanoparticles offer innovative, scaffold-free potentials for tissue engineering: MTE. By using MTE we could generate functional multilayered human VF cell constructs, which can consequently be used to regenerate the voice in patients with VF injuries.

Multimodal virtual histology of rabbit vocal folds by nonlinear microscopy and nano computed tomography.

Human vocal folds (VFs) possess a unique anatomical structure and mechanical properties for human communication. However, VFs are prone to scarring as a consequence of overuse, injury, disease or surgery. Accumulation of scar tissue on VFs inhibits proper phonation and leads to partial or complete loss of voice, with significant consequences for the patient's quality of life. VF regeneration after scarring provides a significant challenge for tissue engineering therapies given the complexity of tissue microarchitecture. To establish an effective animal model for VF injury and scarring, new histological methods are required to visualize the wound repair process of the tissue in its three-dimensional native environment. In this work, we propose the use of a combination of nonlinear microscopy and nanotomography as contrast methods for virtual histology of rabbit VFs. We apply these methods to rabbit VF tissue to demonstrate their use as alternatives to conventional VF histology that may enable future clinical studies of this injury model.

Heterogeneity of Stem Cells in theHuman Vocal Fold Mucosa.

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

Kinematic model for simulating mucosal wave phenomena on vocal folds

Mucosal waves have been found to be important for evaluating vocal fold vibrations in laryngological practice. While they are routinely evaluated visually, the knowledge on the physical phenomena related to mucosal wave propagation is limited. Kymographic imaging, in particular, reveals various mucosal wave features that deserve more understanding in order to advance functional diagnostics of voice disorders. Here, a kinematic model is presented which simulates mucosal waves on human vocal folds. The vocal fold geometry is based on a parametrically adjustable M5 model. A kinematic rule is used for simulating the propagation of the mucosal wave from the bottom of the vocal folds upwards and laterally over the upper vocal fold surface. The model maps the changes of the coronal shape of the vocal folds through vibration cycles. The vibration characteristics including the mucosal wave movements are then visualized using a synthetic kymogram graphically obtained through a local illumination method. The model can serve as an educational and research tool for studying the mucosal wave features and their appearance in laryngeal kymographic images.

Differentiation potential of the cells in the macula flava of the human vocal fold mucosa

The latest research suggests cells in the maculae flavae located at both ends of the lamina propria of the human vocal fold mucosa have stemness. This study investigated the differentiation potential of the cells in the maculae flavae of the human vocal fold mucosa. Four normal human adult vocal folds from surgical specimens were used. After extraction of the anterior maculae flavae located at the anterior end of the lamina propria of the human vocal fold mucosa under microscope, the maculae flavae were minced, cultured and proliferated in mesenchymal stem cell growth medium and morphological features were assessed. Cell surface markers were detected using flow cytometry. Cell differentiation into adipogenic, chondrogenic and osteogenic lineages was performed. Cell’s differentiation potential was assessed using a human pluripotent stem cell functional identification kit and immunohistochemistry. Subcultured cells formed a colony-forming unit. Subcultured cells expressed CD90, CD105 and CD73 and lacked expression of CD45, CD34, CD11b, CD19 and HLA-DR. They differentiated into adipogenic, chondrogenic and osteogenic lineages. Consequently, the cell features in the maculae flavae meet the minimal criteria defining mesenchymal stromal cells. In addition, subcultured cells differentiated into ectoderm, mesoderm and endoderm and expressed stage-specific embryonic antigen 3 (SSEA-3). The results of this study are consistent with the hypothesis that the cells in the maculae flavae in the lamina propria of the human vocal fold mucosa are putative stem cells.

Dilated Minute Chambers in Laryngeal Vocal Fold Polyps: Histopathological and Ultrastructural Features

In Reinke's space of human vocal fold, type III collagen forms a three dimensional network and this contains numerous minute chambers in between these fibers. These compartments are occupied by glycosaminoglycans and glycoproteins. In laryngeal fold lesions, such as Reinke's edema and vocal fold polyps, proteoglycan (PG)/hyaluronic acid (HA) components of extracellular matrix increased. We investigated the size and quantity of the minute chambers within Reinke's space, filled with PG/HA with the aid of transmission electron microscopy. Eight vocal fold polyps and 10 mucosal biopsies (as control group) were all evaluated by light microscopy and electron microscopy. We detected that PG/HA in extracellular matrix had been increased in vocal fold lesions when compared with control group, by Alcian Blue-pH 2.5 stain. The mean volume of the chambers in Reinke's space of normal larynx was measured as 0.040233 µm2 whereas the mean volume of these chambers in vocal fold polyps was measured as 6.420221 µm2. The difference between the volumes of these chambers in vocal fold polyps and in control group was statistically significant (P = 0.001). Within these chambers PG/HA were found and PG/HA filling these chambers were increased in vocal fold polyps. We think proteoglycan and glycosaminoglycans, especially HA, play an important role in determining biochemical properties of vocal fold lesions.

The effects of cytosporone-B, a novel antifibrotic agent, on vocal fold fibroblasts.

Our laboratory recently described NR4A1 as an endogenous inhibitor of TGF-β-induced vocal fold (VF) fibrosis. Our prior report described the temporal expression of NR4A1 during VF healing in vivo and the effects of NR4A1 knockdown on fibroplastic cell activities in vitro. Based on these findings, we hypothesized that cytosporone-B (Csn-B), an NR4A1 agonist, may hold significant therapeutic potential. In vitro. Human VF fibroblasts were exposed to TGF-β1+/-Csn-B. Expression of genes related to fibrosis were quantified. In addition, contraction was assayed as a surrogate for the fibrotic phenotype in our cell line. TGF-B1 stimulated COL1A1 and ACTA2, as expected. Csn-B significantly downregulated TGF-β1-mediated upregulation of these genes (P = .009, P = .03, respectively). Csn-B had no effect on genes related to TGF-β/Smad signaling. Csn-B also decreased the TGF-β1-mediated contractile phenotype in our cells (P = .004). NR4A1 is an endogenous inhibitor of fibrosis in the vocal folds and Csn-B, as an NR4A1 agonist, may evolve as an ideal, therapeutic candidate for this challenging condition. NA Laryngoscope, 128:E425-E428, 2018.

3D multiscale imaging of human vocal folds using synchrotron X-ray microtomography in phase retrieval mode

Human vocal folds possess outstanding abilities to endure large, reversible deformations and to vibrate up to more than thousand cycles per second. This unique performance mainly results from their complex specific 3D and multiscale structure, which is very difficult to investigate experimentally and still presents challenges using either confocal microscopy, MRI or X-ray microtomography in absorption mode. To circumvent these difficulties, we used high-resolution synchrotron X-ray microtomography with phase retrieval and report the first ex vivo 3D images of human vocal-fold tissues at multiple scales. Various relevant descriptors of structure were extracted from the images: geometry of vocal folds at rest or in a stretched phonatory-like position, shape and size of their layered fibrous architectures, orientation, shape and size of the muscle fibres as well as the set of collagen and elastin fibre bundles constituting these layers. The developed methodology opens a promising insight into voice biomechanics, which will allow further assessment of the micromechanics of the vocal folds and their vibratory properties. This will then provide valuable guidelines for the design of new mimetic biomaterials for the next generation of artificial larynges.

Permeability and Weibel-Palade Bodies of the Blood Vessels in the Human Vocal Fold Mucosa.

Transendothelial exchange and permeability of the capillaries in Reinke space (the superficial layer of the lamina propria) of the vocal fold mucosa affect physiological and pathological conditions of the human vocal fold mucosa. The mechanism of permeability and Weibel-Palade bodies of the blood vessels in the human vocal fold mucosa were investigated using electron microscopy. Histologic analysis of the human vocal fold. Six normal human vocal folds (three adults and three newborns) obtained from autopsy cases and three human vocal folds with Reinke edema from surgical specimens were investigated under transmission electron microscopy. There were three possible capillary wall transport systems related to the permeability of the blood vessels in the vocal fold mucosa: 1) Fenestra transport, plasma exuded from the capillaries into surrounding tissue via the fenestration with or without a diaphragm; 2) vesicular transport (transcellular transport via vesicles), the use of vesicles to ferry fluid and solutes across endothelial cells; and 3) junctional transport (intercellular transport), molecules passed through intercellular gaps between endothelial cells. Weibel-Palade bodies were present in the cytoplasm of endothelial cells both in adults and newborns. They were present in high numbers in the cytoplasm of endothelial cells, with intercellular transport in the vocal folds with Reinke edema. There were three types of mechanisms for the permeability of the blood vessels in the human vocal fold mucosa. Some physiologically active substances, such as histamine produced by Weibel-Palade bodies, may adversely influence the permeability of the blood vessels. NA. Laryngoscope, 2588-2592, 2018.

High-Performance Agent-Based Modeling Applied to Vocal Fold Inflammation and Repair.

Fast and accurate computational biology models offer the prospect of accelerating the development of personalized medicine. A tool capable of estimating treatment success can help prevent unnecessary and costly treatments and potential harmful side effects. A novel high-performance Agent-Based Model (ABM) was adopted to simulate and visualize multi-scale complex biological processes arising in vocal fold inflammation and repair. The computational scheme was designed to organize the 3D ABM sub-tasks to fully utilize the resources available on current heterogeneous platforms consisting of multi-core CPUs and many-core GPUs. Subtasks are further parallelized and convolution-based diffusion is used to enhance the performance of the ABM simulation. The scheme was implemented using a client-server protocol allowing the results of each iteration to be analyzed and visualized on the server (i.e., in-situ) while the simulation is running on the same server. The resulting simulation and visualization software enables users to interact with and steer the course of the simulation in real-time as needed. This high-resolution 3D ABM framework was used for a case study of surgical vocal fold injury and repair. The new framework is capable of completing the simulation, visualization and remote result delivery in under 7 s per iteration, where each iteration of the simulation represents 30 min in the real world. The case study model was simulated at the physiological scale of a human vocal fold. This simulation tracks 17 million biological cells as well as a total of 1.7 billion signaling chemical and structural protein data points. The visualization component processes and renders all simulated biological cells and 154 million signaling chemical data points. The proposed high-performance 3D ABM was verified through comparisons with empirical vocal fold data. Representative trends of biomarker predictions in surgically injured vocal folds were observed.

Nonlinear viscoelastic characterization of human vocal fold tissues under large-amplitude oscillatory shear (LAOS)

Viscoelastic shear properties of human vocal fold tissues were previously quantified by the shear moduli (G' and G″). Yet these small-strain linear measures were unable to describe any nonlinear tissue behavior. This study attempted to characterize the nonlinear viscoelastic response of the vocal fold lamina propria under large-amplitude oscillatory shear (LAOS) with a stress decomposition approach. Human vocal fold cover and vocal ligament specimens from eight subjects were subjected to LAOS rheometric testing with a simple-shear rheometer. The empirical total stress response was decomposed into elastic and viscous stress components, based on odd-integer harmonic decomposition approach with Fourier transform. Nonlinear viscoelastic measures derived from the decomposition were plotted in Pipkin space and as rheological fingerprints to observe the onset of nonlinearity and the type of nonlinear behavior. Results showed that both the vocal fold cover and the vocal ligament experienced intercycle strain softening, intracycle strain stiffening, as well as shear thinning both intercycle and intracycle. The vocal ligament appeared to demonstrate an earlier onset of nonlinearity at phonatory frequencies, and higher sensitivity to changes in frequency and strain. In summary, the stress decomposition approach provided much better insights into the nonlinear viscoelastic behavior of the vocal fold lamina propria than the traditional linear measures.