Characterization of chronic vocal fold scarring in a rabbit model

The purpose of the current study was to assess the histologic and rheologic properties of the scarred vocal fold lamina propria during a chronic phase of wound repair in a rabbit model. Eighteen rabbit larynges were scarred using a procedure that involved stripping the vocal fold lamina propria down to the thyroarytenoid muscle, using 3-mm microforceps. The approximate dimension of injury to the vocal fold was 3 x 1.5 x 0.5 mm [length x width x depth]. At 6 months postoperatively, histologic analysis of the scarred and control lamina propria in eight of these rabbits was completed for collagen, procollagen, elastin, and hyaluronic acid. Compared with control samples, scarred tissue samples revealed fragmented and disorganized elastin fibers. Additionally, collagen was significantly increased, organized, and formed thick bundles in the scarred vocal fold lamina propria. Measurements of the viscoelastic shear properties of the scarred and control lamina propria in the remaining 10 rabbits revealed increased elastic shear modulus (G') in 8 of 10 scarred samples and increased dynamic viscosity (eta') in 9 of 10 scarred samples. Although rheologic differences were not statistically significant, they revealed that on average, scarred samples were stiffer and more viscous than the normal controls. Histologic data are interpreted as indicating that by 6 months postinjury, the scarred rabbit vocal fold has reached a mature phase of wound repair, characterized by an increased, organized, and thick bundle collagen matrix. Rheologic data are interpreted as providing support for the potential role of increased, thick bundle collagen, and a disorganized elastin network on shear stiffness and dynamic viscosity in the chronic vocal fold scar. Based on these results, a 6-month postoperative time frame is proposed for future studies of chronic vocal fold scarring using the rabbit animal model.     

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.     

Histologic and Rheologic Characterization of Vocal Fold Scarring

Scarring of the vocal fold causes considerable dysphonia and presents significant treatment challenges. A rabbit model was developed to investigate the histologic ultrastructure and rheologic properties of the scarred vocal fold lamina propria. Eleven rabbit larynges were scarred by means of forcep biopsy. Sixty days postoperatively, the rabbits were sacrificed and their vocal folds were harvested. Histological analysis of the scarred and normal lamina propria was completed for collagen, procollagen, elastin, and hyaluronic acid. Linear viscoelastic shear properties of the tissues were also measured, including elastic shear modulus and dynamic viscosity. Compared to normal vocal fold lamina propria, scarred tissues demonstrated significantly less collagen, an increase in procollagen, and a decrease in elastin. Rheologically, both elastic shear modulus and dynamic viscosity were significantly higher for the scarred tissues. Increased stiffness and viscosity do not appear to result from an increase in collagen, but rather appear to be related to the presence of new, disorganized collagen scaffolding. Results are interpreted in terms of the possible role of interstitial proteins in the etiology of increased stiffness and viscosity, which requires further investigation. This animal model should allow for systematic future investigations of vocal fold scarring and its treatment.     

Current Understanding and Review of the Literature: Vocal Fold Scarring

Vocal fold scarring is the greatest cause of poor voice after vocal fold injury. Scarring causes a disruption of the viscoelastic layered structure of the lamina propria, an increase in stiffness of the vibratory structure, and glottic incompetence. Current treatments for this complex condition are inconsistent and often produce suboptimal results. Research investigating this condition has dramatically increased over the last several years. The literature has been directed toward understanding vocal fold scarring at the biological level and translating this to the clinical forum. We present an up-to-date, thorough, and scholarly review of the literature in vocal fold scarring since 1996.     

A simple-shear rheometer for linear viscoelastic characterization of vocal fold tissues at phonatory frequencies

Previous studies reporting the linear viscoelastic shear properties of the human vocal fold cover or mucosa have been based on torsional rheometry, with measurements limited to low audio frequencies, up to around 80 Hz. This paper describes the design and validation of a custom-built, controlled-strain, linear, simple-shear rheometer system capable of direct empirical measurements of viscoelastic shear properties at phonatory frequencies. A tissue specimen was subjected to simple shear between two parallel, rigid acrylic plates, with a linear motor creating a translational sinusoidal displacement of the specimen via the upper plate, and the lower plate transmitting the harmonic shear force resulting from the viscoelastic response of the specimen. The displacement of the specimen was measured by a linear variable differential transformer whereas the shear force was detected by a piezoelectric transducer. The frequency response characteristics of these system components were assessed by vibration experiments with accelerometers. Measurements of the viscoelastic shear moduli (G' and G") of a standard ANSI S2.21 polyurethane material and those of human vocal fold cover specimens were made, along with estimation of the system signal and noise levels. Preliminary results showed that the rheometer can provide valid and reliable rheometric data of vocal fold lamina propria specimens at frequencies of up to around 250 Hz, well into the phonatory range.     

Interstitial protein alterations in rabbit vocal fold with scar

Fibrous and interstitial proteins compose the extracellular matrix of the vocal fold lamina propria and account for its biomechanic properties. Vocal fold scarring is characterized by altered biomechanical properties, which create dysphonia. Although alterations of the fibrous proteins have been confirmed in the rabbit vocal fold scar, interstitial proteins, which are known to be important in wound repair, have not been investigated to date. Using a rabbit model, interstitial proteins decorin, fibromodulin, and fibronectin were examined immunohistologically, two months postinduction of vocal fold scar by means of forcep biopsy. Significantly decreased decorin and fibromodulin with significantly increased fibronectin characterized scarred vocal fold tissue. The implications of altered interstitial proteins levels and their affect on the fibrous proteins will be discussed in relation to increased vocal fold stiffness and viscosity, which characterizes vocal fold scar.     

The intermediate layer: A morphologic study of the elastin and hyaluronic acid constituents of normal human vocal folds

The lamina propria of vocal folds are important in voice production. We evaluated the morphologic features of elastin and hyaluronic acid, two important constituents of the lamina propria. Thirty normal human vocal folds were obtained from patients dying of traumatic causes without vocal fold injury. These tissues were immediately prepared for histologic and ultrastructural examination by standard methods. For specific study of the ultrastructure of the layers of the lamina propria, six vocal folds were divided horizontally through the midplane of the lamina propria. We found that the elastin composition of the vocal folds is variable, the largest amount being seen in the midportion on elastin-van Gieson (EVG) staining and ultrastructural evaluation. The superficial layer of the lamina propria contains fewer large elastin fibers. In this region, we found that elastin was predominantly composed of elaunin and oxytalan, which stain poorly with EVG. Using computer-assisted image analysis, we quantified the differences in elastin composition between the layers. The amount of elastin varied between men and women, and these differences could not be accurately measured by the methods employed. Hyaluronic acid was abundant especially in the midportion of the lamina propria and was significantly more abundant in men than women on quantification. The significance of these observations in normal vocal folds is discussed.     

Viscoelastic modeling of canine vocalis muscle in relaxation

Analysis of vocal fold vibration requires information on the viscoelastic properties of the vocalis muscle. The force response of two canine vocalis muscles was measured in one-dimensional, stepwise elongation of the tissue as a function of time with a computer-controlled ergometer. The viscoelastic behavior of the muscle in its passive state was demonstrated through the relaxation test. A quasilinear viscoelastic model was used to parametrize the relaxation function, and results are reported for various levels of strain. Furthermore, a model was used to obtain theoretical time-dependent stress-strain curves to compare with experimental data.     

Allograft (Alloderm) and Autograft (Temporalis Fascia) Implantation for Glottic Insufficiency: A Novel Approach

Objective

Traditionally, glottic insufficiency because of scar, atrophy, and sulcus has been treated by injection or medialization laryngoplasty. These procedures do not reestablish the vertical height of the vocal fold margin. We propose soft tissue augmentation laryngoplasty with allograft (sheet Alloderm; LifeCell Corporation, Branchburg, NJ) or autograft (temporalis fascia) via a minithyrotomy or a transoral approach.

Study Design

A retrospective case series analysis of 21 patients treated by sheet Alloderm or temporalis fascia for correction of glottic insufficiency.

Methods

Twenty-one patients with glottic insufficiency secondary to scar, atrophy, or sulcus were treated. Ten failed prior techniques. Seventeen had minithyrotomy by a small fenestration in the thyroid cartilage. Exploration of scar or lamina propria through the fenestration allowed for the creation of a pocket for Alloderm implantation within the intermediate layer of the lamina propria. Four patients underwent a transoral approach by cordotomy with either Alloderm or temporalis fascia implantation, which also allowed for exploration of scar but required repair using sutures. These implantation approaches allowed for both restoration of the layered structure and augmentation of the middle third of the musculomembranous vocal fold. Preoperative and postoperative videostroboscopic examinations were reviewed with review of clinical outcome.

Results

With a median follow-up time of 12 months, patients demonstrated excellent long-term vocal fold augmentation and minimal absorption of the implant in 19 out of 21 patients. There is improved pliability of the vocal fold with good oscillation in scar patients.

Conclusion

Minithyrotomy with soft tissue augmentation is a novel approach for soft tissue augmentation of glottic insufficiency. It has the advantage of augmentation of the medial edge of the vocal fold with a soft tissue implant that has long-term viability. Its role should be explored further in patients with atrophy and scar.     

A constitutive model of the human vocal fold cover for fundamental frequency regulation

The elastic as well as time-dependent mechanical response of the vocal fold cover (epithelium and superficial layer of the lamina propria) under tension is one key variable in regulating the fundamental frequency. This study examines the hyperelastic and time-dependent tensile deformation behavior of a group of human vocal fold cover specimens (six male and five female). The primary goal is to formulate a constitutive model that could describe empirical trends in speaking fundamental frequency with reasonable confidence. The constitutive model for the tissue mechanical behavior consists of a hyperelastic equilibrium network in parallel with an inelastic, time-dependent network and is combined with the ideal string model for phonation. Results showed that hyperelastic and time-dependent parameters of the constitutive model can be related to observed age-related and gender-related differences in speaking fundamental frequency. The implications of these findings on fundamental frequency regulation are described. Limitations of the current constitutive model are discussed.     

Laser Doppler Measurements of the Vocal Fold Blood Micro-Circulation

The authors have developed a laser Doppler method, referred to as LDF+LS (laser Doppler flowmetry by laryngostereometry), for measuring the vocal fold micro-circulation. The vocal folds of 103 patients were examined during general anesthesia. A laser Doppler probe was placed on defined positions at the vocal fold edge: midmembranous position (MP), 2 mm and 4 mm behind, and 1 mm anterior to MP. Three parameters were derived, ie, the concentration (CMBC) and velocity (V) of moving blood cells, and the product of both resulting in perfusion: P = CMBCxV. The results of 53 subjects with normal vocal fold status showed that the V at MP was significantly lower than 2 mm behind MP (P < 0.05). Men had significantly higher velocity than women (P < 0.05). Subjects older than 65 years had higher perfusion and CMBC as compared with younger subjects (P < 0.01). Smokers with normal vocal folds had higher velocity than non-smokers (P < 0.05). Measurements of healthy vocal folds were compared with benign vocal fold pathology. Vocal fold polyps had significantly higher perfusion than normal vocal folds (P < 0.01). In conclusion, the laser Doppler measurement is a sensitive tool estimating superficial blood flow in both normal and pathological vocal folds. The blood flow in normals differs with respect to gender and age probably due to variations in micro-circulation of the mucosa and lamina propria. Vocal fold pathology and external factors such as smoking result in blood flow changes.     

Infraglottic aspect of canine vocal fold vibration: Effect of increase of mean airflow rate and lengthening of vocal fold

The mucosal upheaval (MU), where the mucosal wave starts and propagates upward, appears only when the vocal fold vibrates. The location of the MU histologically and the effect of changes in mean air flow rate (MFR) and vocal fold length on occurrence of the MU were studied in twelve excised canine larynges. The lower surface of the vocal fold was marked to serve as a landmark for subsequent study. Cricothyroid approximation was performed to lengthen the vocal fold. After taking high-speed pictures or recording stroboscopic images from the tracheal side, a small cut wound was made at the mark. This wound served to compare the position of the MU with the histologically identified location of the mark. The larynx was then sectioned in the frontal plane. Before lengthening the vocal fold, the MU occurred on the area where the lamina propria became thinner and where the muscular layer neared the epithelial layer. After lengthening the vocal fold, the MU actually shifted medially compared with its original position. The subglottic area surrounded by the bilateral MUs became longer and thinner. Whether or not complete glottal closure during a vibratory cycle was achieved did not alter these findings. In contrast, with a fixed vocal fold length the MU appeared more laterally as MFR increased, but, based on the relation with the mark, its location on the vocal fold did not change from its original position before increase of MFR.     

Simulated effects of cricothyroid and thyroarytenoid muscle activation on adult-male vocal fold vibration

Adjustments to cricothyroid and thyroarytenoid muscle activation are critical to the control of fundamental frequency and aerodynamic aspects of vocal fold vibration in humans. The aerodynamic and physical effects of these muscles are not well understood and are difficult to study in vivo. Knowledge of the contributions of these two muscles is essential to understanding both normal and disordered voice physiology. In this study, a three-mass model for voice simulation in adult males was used to produce systematic changes to cricothyroid and thyroarytenoid muscle activation levels. Predicted effects on fundamental frequency, aerodynamic quantities, and physical quantities of vocal fold vibration were assessed. Certain combinations of these muscle activations resulted in aerodynamic and physical characteristics of vibration that might increase the mechanical stress placed on the vocal fold tissue.     

Material parameter computation for multi-layered vocal fold models

Today, the prevention and treatment of voice disorders is an ever-increasing health concern. Since many occupations rely on verbal communication, vocal health is necessary just to maintain one's livelihood. Commonly applied models to study vocal fold vibrations and air flow distributions are self sustained physical models of the larynx composed of artificial silicone vocal folds. Choosing appropriate mechanical parameters for these vocal fold models while considering simplifications due to manufacturing restrictions is difficult but crucial for achieving realistic behavior. In the present work, a combination of experimental and numerical approaches to compute material parameters for synthetic vocal fold models is presented. The material parameters are derived from deformation behaviors of excised human larynges. The resulting deformations are used as reference displacements for a tracking functional to be optimized. Material optimization was applied to three-dimensional vocal fold models based on isotropic and transverse-isotropic material laws, considering both a layered model with homogeneous material properties on each layer and an inhomogeneous model. The best results exhibited a transversal-isotropic inhomogeneous (i.e., not producible) model. For the homogeneous model (three layers), the transversal-isotropic material parameters were also computed for each layer yielding deformations similar to the measured human vocal fold deformations.     

Measurements of vocal fold tissue viscoelasticity: approaching the male phonatory frequency range

Viscoelastic shear properties of human vocal fold tissues have been reported previously. However, data have only been obtained at very low frequencies (< or = 15 Hz). This necessitates data extrapolation to the frequency range of phonation based on constitutive modeling and time-temperature superposition. This study attempted to obtain empirical measurements at higher frequencies with the use of a controlled strain torsional rheometer, with a design of directly controlling input strain that introduced significantly smaller system inertial errors compared to controlled stress rheometry. Linear viscoelastic shear properties of the vocal fold mucosa (cover) from 17 canine larynges were quantified at frequencies of up to 50 Hz. Consistent with previous data, results showed that the elastic shear modulus (G'), viscous shear modulus (G"), and damping ratio (zeta) of the vocal fold mucosa were relatively constant across 0.016-50 Hz, whereas the dynamic viscosity (eta') decreased monotonically with frequency. Constitutive characterization of the empirical data by a quasilinear viscoelastic model and a statistical network model demonstrated trends of viscoelastic behavior at higher frequencies generally following those observed at lower frequencies. These findings supported the use of controlled strain rheometry for future investigations of the viscoelasticity of vocal fold tissues and phonosurgical biomaterials at phonatory frequencies.     

Towards the Expression of Sex Hormone Receptors in the Human Vocal Fold

BACKGROUND: The human larynx is assumed to be a steroid receptor target organ. There are only very limited data on the evidence of steroid receptors in the vocal folds, although voice alterations due to hormonal influence and treatment have been found. GOAL OF THE STUDY: To investigate the expression of estrogen alpha, progesterone, and androgen receptors in human vocal folds (vocalis muscle, glands, lamina propria, epithelium). METHODS: Immunohistochemically, vocal fold cadaver specimens of 15 autopsied patients (6 women, 9 men), which were taken approximately 4 to 8 hours postmortem were investigated. Furthermore, one (male) vocal fold biopsy obtained intraoperatively during a laryngectomy was tested. RESULTS: No specific immunohistochemical staining for the different types of steroid hormones investigated could be observed in either the postmortem taken biopsies nor the intraoperatively one. However, several unspecific staining patterns could be observed. CONCLUSION: The results of this study contradict recently published data and question the expression of sex hormone receptors in the vocal folds. Main causes of false interpretations of unspecific staining are discussed.     

Determination of superior surface strains and stresses, and vocal fold contact pressure in a synthetic larynx model using digital image correlation

Stresses and strains within the vocal fold tissue may play a critical role in voice fatigue, in tissue damage and resulting voice disorders, and in tissue healing. In this study, experiments were performed to determine mechanical fields on the superior surface of a self-oscillating physical model of the human vocal folds using a three-dimensional digital image correlation method. Digital images obtained using a high-speed camera together with a mirror system were used to measure displacement fields, from which strains, strain rates, and stresses on the superior surface of the model vocal folds were computed. The dependence of these variables on flow rate was established. A Hertzian impact model was used to estimate the contact pressure on the medial surface from superior surface strains. A tensile stress dominated state was observed on the superior surface, including during collision between the model folds. Collision between the model vocal folds limits the medial-lateral stress levels on the superior surface, in conjunction with compressive stress or contact pressure on the medial surface.     

Simple model for frozen-in viscoelastic stress in optical fibers

We have measured residual stress in a commercial single mode optical fiber and observed its relaxation in an annealing process with a polariscopic stress measurement method. After annealing the fiber for an hour at various high temperatures in a specially made image furnace we have measured the temperature dependent relaxation of frozen-in viscoelastic stress in the fiber. We have proposed a simple physical model to explain recently observed relaxation of frozen-in viscoelastic stress in optical fibers based on the Kelvin-Voigt model of viscoelastic materials. The experimental results are explained with our model of frozen-in viscoelastic stress by introducing simplified four-step procedures for fiber drawing.     

Cooperative Regulation of Vocal Fold Morphology and Stress by the Cricothyroid and Thyroarytenoid Muscles

Voice is produced by vibrations of vocal folds that consist of multiple layers. The portion of the vocal fold tissue that vibrates varies depending primarily on laryngeal muscle activity. The effective depth of tissue vibration should significantly influence the vibrational behavior of the tissue and resulting voice quality. However, thus far, the effect of the activation of individual muscles on the effective depth is not well understood. In this study, a three-dimensional finite element analysis is performed to investigate the effect of the activation of two major laryngeal muscles, the cricothyroid (CT) and thyroarytenoid (TA) muscles, on vocal fold morphology and stress distribution in the tissue. Because structures that bear less stress can easily be deformed and involved in vibration, information on the morphology and stress distribution may provide a useful estimate of the effective depth. The results of the analyses indicate that the two muscles perform distinct roles, which allow cooperative control of the morphology and stress. When the CT muscle is activated, the tip region of the vocal folds becomes thinner and curves upward, resulting in the elevation of the stress magnitude all over the tissue to a certain degree that depends on the stiffness of each layer. On the other hand, the TA muscle acts to suppress the morphological change and controls the stress magnitude in a position-dependent manner. Thus, the present analyses demonstrate quantitative relationships between the two muscles in their cooperative regulation of vocal fold morphology and stress.