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.     

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.     

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.     

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.     

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.     

Investigation of Anti-Hyaluronidase Treatment on Vocal Fold Wound Healing

SUMMARY: Phytochemical constituents of medicinal plants demonstrate inhibition of tissue and bacterial hyaluronidase. Echinacoside is a caffeoyl conjugate of Echinacea with known anti-hyaluronidase properties. The purpose of this study was to investigate the wound healing effects of Echinacea on vocal fold wound healing and functional voice outcomes. Pig animal model. Methods: Vocal fold injury was induced in 18 pigs by unilateral vocal fold stripping. The uninjured vocal fold served as control. Three groups of six pigs randomly received a topical application of 300, 600, or 1,200 mg of standardized Echinacea on the injured side. Animals were euthanized after 3, 10, and 15 days of wound healing. Phonation threshold pressure and vocal economy measurements were obtained from excised larynges. Treatment outcomes were examined by comparing the animals receiving treatment with a set of 19 untreated and 5 historical controls. Treatment effects on wound healing were evaluated by histologic staining for hyaluronan and collagen. Treated larynges revealed improved vocal economy and phonation threshold pressure compared with untreated larynges. Histologically, treated vocal folds revealed stable hyaluronan content and no significant accumulation of collagen compared with control. Findings provide a favorable outcome of anti-hyaluronidase treatment on acute vocal fold wound healing and functional measures of voice.     

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.     

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.     

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.     

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.     

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.     

Spatial arrangement of the structural elements ofvocal fold layers: An adjustment to the vibration process

It is well established that the multilayered structure of the vocal fold is highly adjusted to the requirements of the vibration process during phonation. There is also some partial data indicating that the spatial arrangement of each vocal fold layer corresponds to the functional requirements, and thus facilitate the phonation process. Nevertheless, all reports on the spatial arrangement of the vocal fold structures deal only with an individual element of the vocal fold histologic structure. The present study encompasses the spatial histologic analysis of all major elements of the vocal fold layers. It was demonstrated that the vocal fold epithelial cells, the connective and muscle fibers, and even the blood vessels run parallel to the vocal fold free edge, which indicates a high adjustment to the phonation requirements and the vibration process.     

The Inspiratory Maneuver: A Simple Method to Assess the Superficial Lamina Propria During Endoscopy

We describe a simple means of assessing the condition of the superficial lamina propria during endoscopy, which we use in our practice as an adjunct to careful stroboscopic examination. An intake of breath against partially adducted vocal folds serves to draw the mucosa inferomedially into the glottis, which reveals areas of diminished mobility, the relationship of mucosal lesions to deeper tissues of the vocal folds, and subtle submucosal lesions. This is possible because of the unique geometry of the glottis that renders the vector forces of airflow different at inspiration and expiration. Because the integrity of the superficial lamina propria is essential to healthy phonation, we have found the inspiratory maneuver useful across a wide variety of mucosal pathologic conditions.     

Effects of ambient inhaled ozone on vocal fold mucosa in bonnet monkeys

Deleterious effects of ambient ozone (O₃) on the nasal and lower airways is well documented. However, no investigations have considered possible effects on vocal fold mucosa. To this end, six macaque monkeys were exposed to 0.64 ppm O₃ for 8 h/day for a total of 7 days. Three monkeys were killed 12 h postexposure, and the other three at 7 days postexposure. Larynges were excised and subjected to histological staining and morphometric analyses. Results revealed increases in the thickness of epithelial tissue in the 12 h group. In the 7 day group, differences in both epithelial and lamina propria thicknesses were noted. Additional changes consistent with an initial inflammatory response, with associated disruption in glands and vessels, followed by some evidence of healing, were also noted. Implications of the data for the larynx and voice quality in humans exposed to similar and other environmental toxins are discussed.     

Complications of collagen injection of the vocal fold: report of several unusual cases and review of the literature

Injection laryngoplasty is one of the most frequently performed procedures in patients with voice complaints. Various biomaterials have been used to medialize vocal folds or to treat symptoms of vocal fold scar. The ideal biomaterial would be easily injected through a fine-gauge needle, well tolerated, and long lasting. Injectable collagen preparations fulfill at least two of these criteria, and collagen has been used widely for vocal fold injections. MATERIALS AND METHODS: We present a retrospective review of two unusual complications of collagen injection and a review of the relevant literature on the complications of medical use of collagen compounds. RESULTS: Two patients in whom collagen was injected formed firm submucosal deposits that interrupted the normal mucosal wave and produced significant dysphonia. Surgical removal of these deposits restored the mucosal wave and improved voice quality. Management of this unusual complication of human collagen injection in the vocal fold has not been reported previously. Other complications of collagen injection include hypersensitivity reactions to bovine collagen, local abscess formation at injection sites, and possibly induction of collagen vascular disease in some patients. CONCLUSIONS: Although collagen injections of the vocal fold rarely result in complications, physicians using collagen must be familiar with the types of complications that can occur. Proper diagnosis and prompt management of complications can result in good outcomes.     

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.     

Phonomicrosurgical management of vocal fold polyps: The subepithelial microflap resection technique

Vocal fold polyps are typically caused by acute and chronic trauma to the microvasculature of the superficial lamina propria (SLP). Shearing stresses that are induced by hyperfunctional glottal sound production lead to bleeding into the SLP and malformed neo-vascularized masses. Because the primary process does not involve the epithelium, the authors designed a technique to resect hemorrhagic polyps by epithelial cordotomy with partial or complete preservation of the vocal fold epithelium. This approach is different from the traditional microsurgical resection of hemorrhagic polyps by amputation with or without the carbon dioxide laser. Forty patients who underwent microlaryngoscopic resection of hemorrhagic polyps from 1996 through 1998 were reviewed retrospectively. Thirty-six of the 40 procedures were by epithelial cordotomy and subepithelial removal of the polyp contents. Sixteen of 36 were assisted by a subepithelial infusion of saline and epinephrine, and all were 3 mm to 6 mm. Four of 40 polyps were amputated; all of these were less than 3 mm and were pedicled on a narrow base. Cold instruments were used exclusively in all 40 patients. Postoperative laryngeal stroboscopy within 2 weeks revealed improved mucosal wave propagation and improved glottal closure in all 33 patients in whom postoperative strobovideolaryngoscopy was available. The epithelial cordotomy technique was introduced to minimize disturbance of normal SLP and epithelium. Despite the hemorrhagic nature of these lesions, cold instruments could be used exclusively with facility due to careful microdissection between the polyp and the residual normal SLP and the enhanced hemostasis provided by the subepithelial infusion of saline and epinephrine. The rapid return to improved glottal function is the result of this ultra tissue-sparing technique.