Gender-related differences of hyaluronic acid distribution in the human vocal fold.

OBJECTIVE: To quantify the distribution of hyaluronic acid (HA) within the normal human vocal fold and evaluate gender- and age-related differences. STUDY DESIGN: Prospective, cross-sectional study by age and gender groups using cadaveric vocal folds to study hyaluronic acid differences. METHODS: Twenty-five vocal fold halves obtained at autopsy were evaluated from nine adult male (age range, 34-52 y), seven adult female (age range, 21-41 y), four geriatric male (age range, 65-77 y), and five geriatric female (age range 65-82 y) subjects. Midmembranous vocal fold sections were stained for HA using the Muller-Mowry colloidal iron (acid mucopolysaccharide) stain with and without hyaluronidase. Digital imaging software was used to quantify the distribution of HA across the lamina propria at the vocal fold edge from superficial (subepithelial) to deep (vocalis muscle). RESULTS: A significant gender difference (P =.03) was noted in distribution when controlled for age. Combined, male subjects had a relatively constant distribution pattern throughout the depth of lamina propria. Female subjects showed relatively less HA in the first 15% of depth (most superficial) but showed more HA in the deeper 40% to 100% (toward vocalis muscle) than in male subjects. CONCLUSIONS: Important clinical implications may be extrapolated from our findings. First, relatively less HA in the most superficial area implies less protection from vibratory trauma and overuse and may explain in part why more female than male patients have phonotrauma to phonotraumatic lesions. Second, evaluating the distribution of HA in the normal human vocal fold allows more direction in our application of HA as a therapeutic lamina propria substitute in patients with voice disorders.

Vocal fold proteoglycans and their influence on biomechanics.

OBJECTIVE/HYPOTHESIS: To examine the interstitial proteins of the vocal fold and their influence on the biomechanical properties of that tissue. STUDY DESIGN: Anatomic study of the lamina propria of human cadaveric vocal folds combined with some viscosity testing. METHODS: Identification of proteoglycans is performed with histochemical staining. Quantitative analysis is performed using an image analysis system. A rheometer is used for viscosity testing. Three-dimensional rendering program is used for the computer images. RESULTS: Proteoglycans play an important role in tissue biomechanics. Hyaluronic acid is a key molecule that affects viscosity. DISCUSSION: The proteoglycans of the lamina propria have important biological and biomechanical effects. The role of hyaluronic acid in determining tissue viscosity is emphasized. Viscosity, its effect on phonatory threshold pressure and energy expended due to phonation is discussed. CONCLUSION: Proteoglycans, particularly hyaluronic acid, play important roles in determining biomechanical properties of tissue oscillation. Future research will likely make these proteins of important therapeutic interest.

Analysis of cellular location and concentration in vocal fold lamina propria.

Studies have shown that the lamina propria plays an important role in voice production. Recent studies have analyzed the presence of different proteins and quantified their extent in the lamina propria, but no similar study has yet been done on cellular makeup. The distribution of three different cell types in the lamina propria of 22 human vocal folds was studied. These types are fibroblasts, macrophages, and myofibroblasts. The roles of these cells in the extracellular matrix are described. Their distribution was quantified with use of an image-analysis system. We arbitrarily divided the lamina propria into five sections (each representing 20% of the lamina propria) and compared cell numbers among these sections. Gender comparisons were also made. From these studies it is evident that the cellular distribution in the lamina propria is not uniform. Fibroblasts were more abundant in the deepest 20% of the lamina propria (p < 0.008), myofibroblasts were more abundant in the most superficial 20% (p < 0.016), and in the 36% of our samples that contained macrophages in the lamina propria, there was a significantly higher number of macrophages in the first 20% of the lamina propria (p < 0.003). The only significant gender difference was that women had twice as many macrophages in the most superficial 20% of the lamina propria as men (p < 0.05). The higher myofibroblast activity in the first 20% could indicate that the superficial layer is a region of constant repair. The increased number of macrophages in the superficial layer likely indicates an inflammatory response to inhalants (because of the role of macrophages in the inflammatory response and the fact that only 36% of the patients showed any macrophage activity at all).

Age- and gender-related elastin distribution changes in human vocal folds.

The composition of the lamina propria in human vocal folds has been shown to affect vocal performance. Elastin plays a significant role in the biomechanical effects of the lamina propria. We obtained 19 larynges from the state medical examiner from subjects whose cause of death was unrelated to the trachea and laryngeal regions. The sample contained male and female subjects in the infant, adult, and geriatric age groups. We stained the vocal folds for elastin with Verhoeff's elastic tissue stain and studied them with use of an image analysis system configured for light microscopy. Distributions of elastin were measured from superficial to deep within the lamina propria (from epithelium to vocal muscle). These elastin distributions were then compared with the use of statistical software. The data showed that there was an increase in elastin content from the infant through geriatric stages. No gender-related differences were found. Infant folds had about 23% of the elastin found in adults, and geriatric subjects had about 879% of the elastin found in adults. Both of these results were statistically significant (p < 0.05). The distributions were consistent with previous observations that the lamina propria is a layered structure with most of the elastin present in the intermediate layer. This layer was larger in geriatric subjects than in adult and pediatric subjects. We observed that the fiber diameter appeared to be larger in geriatric subjects (this observation is currently being verified with electron microscopy) whereas smaller, spiraled fibers appeared in pediatric subjects.

Biomechanical and histologic observations of vocal fold fibrous proteins.

This article discusses the molecular composition of the vocal fold and the relationship of fibrous molecules to the biomechanical and physiological performance of the tissue. The components of the extracellular matrix may be divided into fibrous proteins and interstitial proteins. The fibrous proteins, consisting of collagens and elastins, are the focus of this report. Elastin concentration varies by tissue depth in the vocal folds. Variation of elastin by age is reported, but some controversy exists. The biomechanical terms of stress and strain (and stress-strain curves of human vocal folds) are related to the fibrous proteins of the vocal folds. The fibrous proteins, their role in stress, and their effect on the dynamic range of vocal pitch are presented.

Age- and gender-related collagen distribution in human vocal folds.

The composition of the lamina propria in human vocal folds has been shown to affect vocal performance. Collagen plays a significant role in the biomechanical effects of the lamina propria. Specifically, it lends tensile strength to the rapidly oscillating fold. We obtained from a state medical examiner 38 larynges from men and women in infant, adult, and geriatric age groups. We stained the vocal folds for collagen using a picric acid stain and studied them using an image analysis system. Distributions of collagen were measured from the superficial to deep layers (from epithelium to vocalis muscle) within the lamina propria. The data showed an increase in collagen content from infant to adult stages. Infant folds had about 51% of the collagen found in all adults and in geriatric patients (p < .001). There was no significant difference between adult and geriatric folds (p < .16). There was, however, a gender difference in the amount of collagen in both adult and geriatric specimens. Female adult and geriatric folds had about 59% of the collagen found in male adult and geriatric folds (p < .001). The distribution pattern of collagen showed that most of the collagen was present in the deep layer. From these data we conclude that there are age-related and gender-related differences between male and female infant, adult, and geriatric vocal folds. Stress-strain performance studies need to be correlated with histologic findings to better study the phonetic implications of these findings.

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.