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.     

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.     

Numerical analysis and comparison of flow fields in normal larynx and larynx with unilateral vocal fold paralysis

In this study, laryngeal flow fields are investigated and compared in normal larynx and models of larynx with unilateral vocal fold paralysis (UVFP). In paralytic models, three fixed initial glottal gaps are considered to understand the positive or probable negative impacts of surgical operation on unilaterally paralytic larynx, by which the paralyzed vocal fold is brought closer to the mid-plane. Various features of the flow fields have been discussed in detail including glottal gap width, glottal flow rate, glottal exit pressure pattern and glottal jet evolution. The numerical solution of fluid-structure interaction is carried out using ANSYS, and the results confirm some of the favorable effects of surgery on the patient’s larynx. It is also shown that by tightening the glottal gap, some of the problems caused by the presence of a motionless vocal fold, such as leakage through glottal gap in the closure phase resulting in breathy voice can be moderated, although some of the symptoms of this disorder remain relatively unchanged.     

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.     

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.     

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.     

Mechanotransduction of vocal fold fibroblasts and mesenchymal stromal cells in the context of the vocal fold mechanome

The design of cell-based therapies for vocal fold tissue engineering requires an understanding of how cells adapt to the dynamic mechanical forces found in the larynx. Our objective was to compare mechanotransductive processes in therapeutic cell candidates (mesenchymal stromal cells from adipose tissue and bone marrow, AT-MSC and BM-MSC) to native cells (vocal fold fibroblasts-VFF) in the context of vibratory strain. A bioreactor was used to expose VFF, AT-MSC, and BM-MSC to axial tensile strain and vibration at human physiological levels. Microarray, an empirical Bayes statistical approach, and geneset enrichment analysis were used to identify significant mechanotransductive pathways associated with the three cell types and three mechanical conditions. Two databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes) were used for enrichment analyses. VFF shared more mechanotransductive pathways with BM-MSC than with AT-MSC. Gene expression that appeared to distinguish the vibratory strain condition from polystyrene condition for these two cells types related to integrin activation, focal adhesions, and lamellipodia activity, suggesting that vibratory strain may be associated with cytoarchitectural rearrangement, cell reorientation, and extracellular matrix remodeling. In response to vibration and tensile stress, BM-MSC better mimicked VFF mechanotransduction than AT-MSC, providing support for the consideration of BM-MSC as a cell therapy for vocal fold tissue engineering. Future research is needed to better understand the sorts of physical adaptations that are afforded to vocal fold tissue as a result of focal adhesions, integrins, and lamellipodia, and how these adaptations could be exploited for tissue engineering.     

Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses

Criteria are outlined for the design of a bioreactor that can simulate the vibrational stresses in vocal fold movement during speech. Requirements are 0-1 mm amplitudes in the 20-200 Hz frequency range, a variable on-off stress regime, and maintenance of tissue viability over several days. The bioreactor uses dual drivers, one for low frequency (or static) strains, and another for high-frequencies vibrational strains. Response is linear at the driving end for an input of 0-5 V. The amplitude decreases linearly with frequency at constant input voltage, and the phase changes by nearly 180 degrees over the 20-200 Hz range. Human vocal fold fibroblasts were cultured in a polymer substrate and subjected to static and vibrational forces. The results indicate that vibratory strain alters the expression levels of many extracellular matrix-related genes, as well as the spatial distribution of cells and matrix.     

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.     

The postnatal ontogeny of the sexually dimorphic vocal apparatus in goitred gazelles (Gazella subgutturosa)

This study quantitatively documents the progressive development of sexual dimorphism of the vocal organs along the ontogeny of the goitred gazelle (Gazella subgutturosa). The major, male‐specific secondary sexual features, of vocal anatomy in goitred gazelle are an enlarged larynx and a marked laryngeal descent. These features appear to have evolved by sexual selection and may serve as a model for similar events in male humans. Sexual dimorphism of larynx size and larynx position in adult goitred gazelles is more pronounced than in humans, whereas the vocal anatomy of neonate goitred gazelles does not differ between sexes. This study examines the vocal anatomy of 19 (11 male, 8 female) goitred gazelle specimens across three age‐classes, that is, neonates, subadults and mature adults. The postnatal ontogenetic development of the vocal organs up to their respective end states takes considerably longer in males than in females. Both sexes share the same features of vocal morphology but differences emerge in the course of ontogeny, ultimately resulting in the pronounced sexual dimorphism of the vocal apparatus in adults. The main differences comprise larynx size, vocal fold length, vocal tract length, and mobility of the larynx. The resilience of the thyrohyoid ligament and the pharynx, including the soft palate, and the length changes during contraction and relaxation of the extrinsic laryngeal muscles play a decisive role in the mobility of the larynx in both sexes but to substantially different degrees in adult females and males. Goitred gazelles are born with an undescended larynx and, therefore, larynx descent has to develop in the course of ontogeny. This might result from a trade‐off between natural selection and sexual selection requiring a temporal separation of different laryngeal functions at birth and shortly after from those later in life. J. Morphol. 277:826–844, 2016. © 2016 Wiley Periodicals, Inc.     

Tunable mechanical stability and deformation response of a resilin-based elastomer

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues, and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this Article, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G' can be easily tuned within the range of 500 Pa to 10 kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by nonmechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicating elastic modulus values from 200 to 2500 Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled to their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.     

Vocal fold composition and early glottic carcinoma infiltration

ABSTRACT: BACKGROUND: Current imaging techniques provide only limited information pertaining to the extent of infiltration of laryngeal carcinomas into vocal fold tissue layers. Therefore, it is needed to seek the contribute to the body of knowledge surrounding examination and characterization in laryngeal carcinoma infiltration. METHODS: Excised larynges were collected from 30 male laryngectomy patients with an average age of 43.5 years (ranging 36 to 55 years) and history of smoking ([GREATER-THAN OR EQUAL TO]10 years) exhibiting T1, T2, or subglottal (normal vocal fold) carcinomas. Vocal folds were preserved via freezing or immersion in paraffin. The depth of the mucosa, submucosa, and muscular layers in both normal vocal folds and tumor tissues of afflicted vocal folds was measured. RESULTS: The average depths of the mucosa, submucosa, and muscular layers in normal vocal folds were 0.15 [PLUS-MINUS SIGN] 0.06 mm, 2.30 [PLUS-MINUS SIGN] 0.59 mm, and 2.87 [PLUS-MINUS SIGN] 0.88 mm, respectively. Infiltration measurements of T1 tumors showed a depth of 1.62 [PLUS-MINUS SIGN] 0.51 mm and 1.32 [PLUS-MINUS SIGN] 0.49 mm in frozen sections and paraffin-embedded samples, respectively. Similarly, T2 tumors showed a depth of 2.87 [PLUS-MINUS SIGN] 0.68 mm and 2.58 [PLUS-MINUS SIGN] 0.67 mm in frozen sections and paraffin-embedded samples, respectively. T1 and T2 tumors occupied 24.8 [PLUS-MINUS SIGN] 10 and 48.5 [PLUS-MINUS SIGN] 15 percent of the normal vocal fold depth, respectively. CONCLUSION: This data provides a baseline for estimating infiltration of laryngeal carcinomas in vocal fold tissue layers, of particular interest to surgeons. This information may be used to assess typical depths of infiltration, thus allowing for more appropriate selection of surgical procedures based on individual patient assessment.     

Predicting Achievable Fundamental Frequency Ranges in Vocalization Across Species

Vocal folds are used as sound sources in various species, but it is unknown how vocal fold morphologies are optimized for different acoustic objectives. Here we identify two main variables affecting range of vocal fold vibration frequency, namely vocal fold elongation and tissue fiber stress. A simple vibrating string model is used to predict fundamental frequency ranges across species of different vocal fold sizes. While average fundamental frequency is predominantly determined by vocal fold length (larynx size), range of fundamental frequency is facilitated by (1) laryngeal muscles that control elongation and by (2) nonlinearity in tissue fiber tension. One adaptation that would increase fundamental frequency range is greater freedom in joint rotation or gliding of two cartilages (thyroid and cricoid), so that vocal fold length change is maximized. Alternatively, tissue layers can develop to bear a disproportionate fiber tension (i.e., a ligament with high density collagen fibers), increasing the fundamental frequency range and thereby vocal versatility. The range of fundamental frequency across species is thus not simply one-dimensional, but can be conceptualized as the dependent variable in a multi-dimensional morphospace. In humans, this could allow for variations that could be clinically important for voice therapy and vocal fold repair. Alternative solutions could also have importance in vocal training for singing and other highly-skilled vocalizations.     

In reference to phonation larynx fixation: computer graphic record

The vocal apparatus serves phonation. It represents a biocybernetic self-regulating system, disposing of a feedback network of the central nervous system. The larynx is a self-induced vibrating system. The larynx, functioning as the phonation apparatus of the vocal apparatus, is a source of human voice. In every individual its frequency range corresponds to about eight semitones in speech and about two octaves of the so-called chest register in singing, denoted also as a thoracic or modal voice. This is followed by one more octave of the so-called cranial register or falsetto voice. We were interested in changes of the larynx positions at intonation in the fundamental singing registers, both modal and falsetto, in professional male singers. At our disposal were 11 professional male singers. We investigated changes in the position of the laryngeal structures simultaneously with the aid of an X-ray apparatus, the acoustic and mechanical signals registered by means of the B & K 4369 acceleration recorder. It has been found that at phonation with the modal voice a change in the position of the laryngeal structures takes place in two different ways, whereas the larynx movements at falsetto remain the same. It has been suggested that a complex fixation apparatus participates in the phonation larynx movements. Of the same complex character are also the problems connected with the examination of the entire vocal apparatus. For the purpose of compiling the present pieces of knowledge in the field of human voice studies, we have made the most advantageous use of the presently most complex system Authorware for the production of some interactive multimedial programmes on personal computers.     

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.     

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.     

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.     

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.     

Parelectric spectroscopy for noninvasive diagnosis of laryngeal tissue]

Under the influence of an external electrical field, every biological tissue displays characteristic parelectric properties that can be recorded by radiofrequency spectroscopy in a noninvasive contact mode. Parelectric spectroscopy was investigated for its utility as a complementary noninvasive diagnostic procedure in examinations of the larynx, in particular in terms of its ability to differentiate tissue properties. Parelectric spectroscopy was performed in 10 patients submitted to surgical ablation of vocal cord neoplasia under local or insufflation anaesthesia. Measurements were obtained in the area of the neoplasia, and in macroscopically normal tissue in the corresponding vocal cord. In all cases, intra-individual comparison with normal vocal cord tissue revealed lower dipole density and reduced mobility of the affected vocal cord. In addition, the difference between normal and pathological tissue in terms of the parelectric parameters increased with age. The absolute values of dipolar density and mobility revealed no tendency to correlate with different kinds of vocal cord neoplasia. Parelectric spectroscopy may be a useful additional diagnostic tool for monitoring the course of epithelial changes in the larynx.     

The response of vocal fold fibroblasts and mesenchymal stromal cells to vibration

Illumination of cellular changes caused by mechanical forces present within the laryngeal microenvironment may well guide strategies for tissue engineering the vocal fold lamina propria. The purpose of this study was to compare the response of human vocal fold fibroblasts (hVFF) and bone marrow mesenchymal stem cells (BM-MSC) to vibratory stimulus. In order to study these effects, a bioreactor capable of vibrating two cell seeded substrates was developed. The cell seeded substrates contact each other as a result of the sinusoidal frequency, producing a motion similar to the movement of true vocal folds. Utilizing this bioreactor, hVFF and BM-MSC were subjected to 200 Hz vibration and 20% strain for 8 hours. Immunohistochemistry (Ki-67 and TUNEL) was performed to examine cell proliferation and apoptosis respectively, while semi-quantitative RT-PCR was used to assess extracellular matrix related gene expression. HVFF significantly proliferated (p = 0.011) when subjected to 200 Hz vibration and 20% strain, while BM-MSC did not (p = 1.0). A statistically significant increase in apoptosis of BM-MSC (p = 0.0402) was observed under the experimental conditions; however high cell viability (96%) was maintained. HVFF did not have significantly altered apoptosis (p = 0.7849) when subjected to vibration and strain. Semi-quantitative RT-PCR results show no significant differences in expression levels of collagen I (BM-MSC p = 0.1951, hVFF p = v0.3629), fibronectin (BM-MSC p = 0.1951, hVFF p = 0.2513), and TGF-β1 (BM-MSC p = 0.2534, hVFF p = 0.6029) between vibratory and static conditions in either cell type. Finally, smooth muscle actin mRNA was not present in either vibrated or static samples, indicating that no myofibroblast differentiation occurred for either cell type. Together, these results demonstrate that BM-MSC may be a suitable alternative to hVFF for vocal fold tissue engineering. Further investigation into a larger number of gene markers, protein levels, increased number of donors and vibratory conditions are warranted.