Elastic cartilage properties of the tip of the vocal process of the arytenoid cartilage

Abstract

Objectives

This study aimed to investigate the histological and ultrastructural features of the elastic cartilage at the tip of the vocal process in the arytenoid cartilage, which is essential for laryngeal biomechanics.

Methods

Five larynges, including the vocal folds and epiglottis, were examined using transmission electron microscopy. The elastic cartilage at the tip of the vocal process was compared to the epiglottic cartilage within the same larynx to elucidate structural differences.

Results

The elastic cartilage at the vocal process tip consisted of chondrocytes, elastic fibers, collagen fibrils, and ground substances. Chondrocytes in the vocal process were smaller and not distinctly isolated in cartilage cavities (lacunae), unlike those in the epiglottis. Cytoplasmic analysis showed abundant free ribosomes and well‐developed granular endoplasmic reticula, often with dilated cisternae. Amorphous materials were synthesized at the periphery of the cytoplasm and secreted through vesicles. The cartilage matrices were composed of elastic fibers, collagen fibrils, microfibrils, and proteoglycans. The density of elastic fibers was notably higher where elaunin fibers predominated. Collagen fibrils were thin, randomly oriented, and did not form thick bundles. Microfibrils formed delicate three‐dimensional networks with other extracellular matrix components. And proteoglycan granules were located in the interstitial spaces among other cartilage matrix components and attached to them.

Conclusions

The unique structural features of the elastic cartilage at the tip of vocal process, when compared to the epiglottic cartilage, suggest a specialized functional role in maintaining the flexibility and resilience required for vocal fold adduction and abduction during phonation and other laryngeal functions.

Level of evidence: NA

The unique structural features of the elastic cartilage at the tip of vocal process, when compared to the epiglottic cartilage within the same larynx, suggest a specialized functional role in maintaining the flexibility and resilience required for vocal fold adduction and abduction during phonation and other laryngeal functions.

1. INTRODUCTION

The bilateral vocal processes of the arytenoid cartilage are integral to the dynamic formation of the glottis during respiration, swallowing, and phonation. ¹ , ²

Among the laryngeal cartilages, the arytenoid cartilage is unique in comprising two distinct types; hyaline and elastic cartilages. These two types of cartilages differ significantly in both structure and properties. Hyaline cartilage is firm, serving as a structural framework for the larynx, whereas elastic cartilage has flexibility and resilience, accommodating movement.

Previous studies of the vocal process of the arytenoid cartilage using light and electron microscopy have demonstrated the critical role of the elastic cartilage portion of the vocal process for proper physiological functions of the arytenoid cartilage. ³ , ⁴ However, the specific characteristics and significance of the elastic cartilage located at the tip of the vocal process of the arytenoid cartilage remains controversial. In addition, when we consider a distinct functional role of the elastic cartilage located at the tip of the vocal process in laryngeal biomechanics, whether it has the typical architecture of conventional elastic cartilage or not is unknown and of great importance.

In this study, we aim to further clarify the properties of the elastic cartilage at the tip of the vocal process of the arytenoid cartilage of the larynx and explore its functional histoanatomy and physiological significance.

2. MATERIALS AND METHODS

The authors confirm that all procedures conducted in this study adhered to the ethical standards established by the relevant national and institutional guidelines for human experimentation, as approved by the Ethics Committee of Kurume University (Permission Number: 21129). Informed consents were obtained.

Five normal human adult larynges (46.4 ± 9.1 years, mean ± SD, 3 males and 2 females), including the vocal folds and epiglottis, obtained from autopsy cases were investigated. No pathological conditions affecting the tissues of the vocal folds or epiglottis were identified.

2.1. Transmission electron microscopy

The human adult laryngeal specimens (vocal folds and epiglottis) were fixed in 2.5% glutaraldehyde, subsequently rinsed with cacodylate buffer solution and postfixed in 2% osmium tetroxide with a cacodylate buffer solution. Following fixation, the specimens were dehydrated through a series of graded ethanol concentrations and embedded in epoxy resin. Thin sections were prepared using an ultramicrotome and stained with uranyl acetate and lead citrate and tannic acid. Observation was performed using a H‐7650 (HITACHI, Japan) transmission electron microscope (TEM).

2.2. Comparison of elastic cartilage at the tip of the vocal process and the epiglottic cartilage

Among the laryngeal cartilages, only the tip of the vocal process and the epiglottic cartilage are composed of elastic cartilage.

For the same larynx, TEM was utilized to examine and compare the differences between the cell and extracellular matrix characteristics of the epiglottic cartilage and the elastic cartilage at the tip of the vocal process.

The dimensions of chondrocytes and the territorial matrix including cartilage cavities (lacunae) were compared between the tip of vocal process and the epiglottic cartilage. The average sizes were compared statistically (unpaired t test).

3. RESULTS

3.1. Transmission electron microscopic findings of the elastic cartilage in the epiglottis

The chondrocytes of the epiglottic cartilage were situated in cartilage cavities (lacunae) (Figure 1). These cells appeared round to oval with a multiangular surface, with dimensions of 25.6 ± 1.6 μm × 15.2 ± 1.5 μm (mean ± SD). The cell surfaces exhibited scalloped features with short, irregular ridges that protruded into the cartilage cavities (lacunae). In the pericellular region, delicate collagen fibrils were less densely arranged. The cytoplasm of the chondrocytes contained glycogen particles. Mitochondria were small and scarce.

FIGURE 1.

Chondrocyte of epiglottic cartilage (tannic acid staining). Chondrocytes were situated in a cartilage cavity (lacuna). The chondrocyte and cartilage cavity (lacuna) were surrounded by a territorial matrix. The cartilage matrices were comprised of elastic fibers, collagen fibers and proteoglycans, with a predominant presence of frequently branching elastic fibers.

The chondrocytes and cartilage cavities (lacunae) were surrounded by a territorial matrix (Figure 1). The combined size of the territorial matrix and cartilage cavities (lacunae) was 36.6 ± 1.0 μm × 26.6 ± 1.0 μm (mean ± SD). A gradient of increasing fiber diameter and density was observed from the cartilage cavities (lacunae) toward the interterritorial matrix, forming the territorial matrix.

The cartilage matrices were comprised of elastic fibers, collagen fibers, and proteoglycans, with a predominant presence of frequently branching elastic fibers (Figure 1).

3.2. Transmission electron microscopic findings of the chondrocytes in the elastic cartilage at the tip of the vocal process of the arytenoid cartilage

The tip of the vocal process (elastic cartilage) of the arytenoid cartilage was composed of chondrocytes and cartilage matrices (Figures 2 and 3). The tip of the vocal process was connected anteriorly to the posterior macula flava of the vocal fold. The transition between the tip of the vocal process and the posterior macula flava was gradual and the border between them was not clearly delineated.

FIGURE 2.

Chondrocyte at the tip of the vocal process (uranyl acetate and lead citrate staining). The chondrocytes was not distinctly isolated within a small cartilage cavity (lacuna). They were significantly smaller than those of chondrocytes in the epiglottic cartilage. The cartilage cavity and distinct territorial matrix were also significantly smaller than the corresponding structures in the epiglottic cartilage.

FIGURE 3.

Chondrocyte at the tip of the vocal process (tannic acid staining). The cytoplasm of the chondrocyte contains glycogen particles and a lipid droplet.

Chondrocytes at the tip of the vocal process of the arytenoid cartilage were not distinctly isolated within small cartilage cavities (lacunae) (Figure 2). Their average dimensions were 7.9 ± 0.9 μm × 5.4 ± 0.4 μm (mean ± SD), significantly smaller than those of the chondrocytes in the epiglottic cartilage (p < .01). The surfaces of these cells were similarly scalloped, with short irregular ridges extending into the cartilage cavities (lacunae). The territorial matrix was distinct and not wide (Figure 2). The territorial matrix and cartilage cavities (lacunae) measured 8.2 ± 0.9 μm × 5.7 ± 0.4 μm (mean ± SD), also significantly smaller than the corresponding structures in the epiglottic cartilage (p < .01).

The cytoplasm of the chondrocytes contained glycogen particles and lipid droplets (Figure 3). Abundant free ribosomes were observed alongside well‐developed granular endoplasmic reticula, often featuring dilated cisternae, indicating active protein synthesis (Figure 4). Mitochondria were small, and both isolated and aggregated cytoplasmic filaments were present (Figure 4).

FIGURE 4.

Chondrocyte at the tip of the vocal process (uranyl acetate and lead citrate staining). (B) Square region in (A). (C) Square region in (A). Abundant free ribosomes were observed, alongside well‐developed granular endoplasmic reticula, often featuring dilated cisternae.

Coated vesicles were present throughout the cytoplasm and in association with the cell membranes (Figure 5). At the periphery of the cytoplasm, amorphous materials were synthesized and secreted from the vesicles (Figure 5).

FIGURE 5.

Synthesized and secreted amorphous materials from the vesicles of a chondrocyte (uranyl acetate and lead citrate staining). Coated vesicles were present throughout the cytoplasm and in association with the cell membrane. At the periphery of the cytoplasm, amorphous materials were synthesized and secreted from the vesicles.

3.3. Transmission electron microscopic findings of the cartilage matrices in the elastic cartilage at the tip of the vocal process of the arytenoid cartilage

The cartilage matrices were composed of elastic fibers, collagen fibrils, microfibrils, and proteoglycans. Much of the matrix substance consisted of frequently branching elastic fibers (Figure 3).

The density of elastic fibers was notably higher at the tip of the vocal process, where elaunin fibers predominated (Figure 6). Elaunin fibers are elastic‐related fibers, characterized by their distinct fibrillary component (microfibrils) and exhibit a lower abundance of elastic component (elastin, which stains black with tannic acid) compared to typical elastic fibers (Figure 6).

FIGURE 6.

Elaunin fibers (elastic‐related fibers) (tannic acid staining). Elaunin fibers are characterized by their distinct fibrillary component (microfibrils) and exhibited a lower abundance of elastic component (elastin, which stained black with tannic acid) compared to typical elastic fibers.

Collagen fibrils were relatively thin (approximately 40 nm in diameter) and ran in various directions without forming thick bundles (Figure 6).

Microfibrils were distributed throughout the cartilage matrices (Figure 7). They were slender and approximately 10 nm in diameter. Some microfibrils were observed in isolation, whereas others were closely associated with other extracellular matrix components, such as collagen fibrils, elastic fibers, and proteoglycans. The microfibrils formed delicate three‐dimensional networks with other extracellular matrix components.

FIGURE 7.

Collagen fibrils and microfibrils (tannic acid staining). Collagen fibrils were relatively thin and ran in various directions without forming thick bundles. Microfibrils were slender and formed delicate three‐dimensional networks with other extracellular matrix components.

Proteoglycan granules, consisting of proteoglycan particles associated with filaments, were located in the interstitial spaces among other cartilage matrix components (e.g., collagen fibrils, elastic fibers, and microfibrils) and attached to them (Figure 8).

FIGURE 8.

Proteoglycan granules (tannic acid staining). Proteoglycan granules, consisting of proteoglycan particles associated with filaments, were located in the interstitial spaces among other cartilage matrix components and attached to them.

4. DISCUSSION

The laryngeal cartilages consist mainly of the thyroid, cricoid, aryntenoid, and epiglottic cartilages. Among these, the thyroid and cricoid cartilages are made of hyaline cartilage, which is firm and provides structural support to the larynx. The epiglottic cartilage, in contrast, is composed of elastic cartilage, which has flexibility and resilience. On the other hand, the arytenoid cartilage contains both hyaline and elastic cartilage, and the varying properties of these two types make their distribution within the arytenoid particularly significant in terms of their physiological and functional roles.

4.1. Physiological and functional roles of the tip of the vocal process of the arytenoid cartilage

It is generally accepted that the tip of the vocal process of the arytenoid cartilage consists of elastic cartilage. ⁵ However, our study, which utilized whole‐organ serial sectioning, demonstrated that elastic cartilage is present not only at the tip of the vocal process but also extends throughout the superior portion of the arytenoid cartilage, from the vocal process to the apex (Figure 9). ² , ³

FIGURE 9.

Distribution and behavior of elastic cartilage in the vocal process of the arytenoid cartilage (cited from Sato et al. ³ ). Elastic cartilage (shaded) is present not only at the tip of the vocal process but also extends throughout the superior portion of the arytenoid cartilage, from the vocal process to the apex. During adduction and abduction, the vocal process bends specifically at the elastic cartilage region of the vocal process. During adduction, the arytenoids primarily make contact at their superior portion, which is composed of elastic cartilage.

Sato et al. reported that during adduction and abduction, the vocal process bends specifically at the elastic cartilage region of the vocal process (Figure 9). ² , ³ Additionally, the arytenoids primarily make contact at their superior portion, which is composed of elastic cartilage (Figure 9). ² , ³ From a physiological and functional perspective, the presence of elastic cartilage at the tip of the vocal process likely enhances flexibility and resilience during movement of the vocal process during adduction and abduction, whereas the elastic cartilage distributed in the superior portion of the arytenoids serves as a cushion, protecting both the arytenoids and covering mucosa from mechanical stress, which may be caused when the arytenoids come into contact. ² , ³ Furthermore, unlike hyaline cartilage, elastic cartilage does not ossify, allowing it to maintain its functional role throughout a person's lifetime. ² , ³

4.2. Elastic cartilage properties in the tip of the vocal process of the arytenoid cartilage

This study investigated the structural properties of the elastic cartilage at the tip of the vocal process and compared them with the epiglottic elastic cartilage within the same larynx. The results revealed that the elastic cartilage at the tip of the vocal processes exhibit specialized properties that diverge from the typical architecture of conventional elastic cartilage, suggesting a distinct functional role in laryngeal biomechanics.

At the tip of the vocal process, the chondrocytes located within small cartilage cavities (lacunae) were notably smaller and exhibited a higher density compared to those in the epiglottic elastic cartilage. From a functional histoanatomical perspective, a high density of smaller chondrocytes allows pliability, facilitating the flexible movement of the tip of the vocal process.

In the cytoplasm of the chondrocytes, abundant free ribosomes were observed alongside well‐developed granular endoplasmic reticula, often featuring dilated cisternae, indicating active protein synthesis. These chondrocytes were actively engaged in synthesizing and secreting amorphous materials from the vesicles, contributing to the formation of cartilage matrices, including elastic fibers, collagen fibrils, microfibrils, and proteoglycans.

At the tip of the vocal process, fibrous proteins, such as elastic fibers, collagen fibrils, and microfibrils were dense. Elastic fibers were abundant, with elaunin fibers predominating. Elaunin fibers, a form of elastic fiber containing a small amount of elastin, are associated with mechanical resistance, providing structural support. ⁶ , ⁷ The collagen fibrils were thin and arranged in multiple directions without forming thick bundles, whereas microfibrils distributed throughout the cartilage matrices formed a delicate three‐dimensional network with other extracellular matrix components. Proteoglycan granules were observed in the interstitial spaces, interacting with other cartilage matrix components. These extracellular matrix components formed intricate three‐dimensional networks, reinforcing the pliability, resilience, and structural integrity of the tip of the vocal process.

During vocal fold abduction and adduction, the tip of the vocal process bends at the elastic cartilage portion, enduring significant mechanical stress. ² , ³ The extracellular matrix composition at this location suggests that it provides mechanical resistance and structural support, allowing for both flexibility and strength at the tip of the vocal process.

4.3. Unique histological structures around the membranous portion of the vocal fold and tip of the vocal process

Recent investigations have supported the hypothesis that tissue stem cells reside in the anterior and posterior maculae flavae located at both ends of the membranous portion of the human vocal fold, functioning as a stem cell niche, which is a microenvironment nurturing tissue stem cells. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ These findings align with the hypothesis that these tissue stem cells contribute to the growth, development, and aging of the human vocal fold mucosa, ¹⁸ , ¹⁹ , ²⁰ helping maintain the layered structure critical for its function as a vibrating tissue. ²¹ , ²²

The posterior macula flava is attached posteriorly to the tip of the vocal process, and the transition between the posterior macula flava and the tip of the vocal process (elastic cartilage portion) is gradual, with no distinct boundary. ² , ³ This gradual transition in stiffness between the membranous vocal fold and the vocal process likely helps absorb mechanical shock during phonation, protecting the tip of the vocal process from mechanical damage, and enabling it to bend easily during vocal fold adduction and abduction. ⁴

The cells isolated from the maculae flavae of the human vocal fold have demonstrated multipotency, differentiating into adipocytes, osteoblasts, and chondroblasts in vitro. ¹² This suggests that the tissue stem cells within the posterior macula flava may differentiate into chondrocytes in the elastic cartilage at the tip of the vocal process, contributing to the continuous regeneration of chondrocytes. This regenerative capacity may be integral to maintaining the characteristic properties of the elastic cartilage in this region, ensuring the long‐term functionality of the vocal process.

4.4. Study limitations and future directions

Although this study provides novel insights into the structural properties of the tip of the vocal process using transmission electron microscopy, the scope is limited by the focus on histoanatomical observation. Further investigations employing complementary techniques are necessary to validate the proposed roles of elastic cartilage in the vocal process dynamics.

5. CONCLUSIONS

The unique histological and ultrastructural features of elastic cartilage at the tip of vocal process underscore its specialized role in laryngeal biomechanics. These findings suggest that the elastic cartilage in the tip of the vocal process plays a critical function in maintaining the flexibility and mechanical resilience required for vocal fold adduction and abduction, particularly during phonation.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Sato K, Chitose S, Sato K, Sato F, Ono T, Umeno H. Elastic cartilage properties of the tip of the vocal process of the arytenoid cartilage. Laryngoscope Investigative Otolaryngology. 2025;10(1):e70071. doi: 10.1002/lio2.70071