REVIEW URRENT C OPINION
Histological changes in vocal fold growth and aging Maggie A. Kuhn
Purpose of review Sophisticated descriptions of the highly specialized vocal fold microarchitecture have been available for over three decades, but how this anatomy evolves with growth and aging remains an area of active investigation and, at times, a source of controversy. As our aging population expands and interest in pediatric voice disorders blossoms, it is timely to consider our contemporary understanding of evolving vocal fold histology and its implications for voice production. Recent findings Novel applications of existing and emerging biotechnology, development of animal models and skillful use of human specimens have afforded greater insights into the histologic vocal fold changes seen throughout the lifespan in health and disease. Summary Burgeoning knowledge has laid the foundation for more comprehensive models of vocal fold histology and has led to the development of innovative therapies for challenging voice disorders. Keywords aging, development, extracellular matrix, lamina propria, vocal fold histology
INTRODUCTION The specialized microanatomy of the vocal folds is essential for the complex mechanism of voice production. Intimate knowledge of vocal fold histology is fundamental for understanding voice disorders and necessary for approaching treatment. This review begins with a discussion of the most recent expansion of our knowledge regarding normal adult vocal fold histology. As with much of the human anatomy, the vocal folds’ histologic features transform during development and throughout the lifespan. Some changes are a consequence of normal growth and aging, whereas others represent disease states. Recent contributions to our growing understanding of these changes will be addressed. Lastly, the development of promising, novel therapeutic interventions on the basis of our understanding of vocal fold histophysiology will be reviewed.
NORMAL ADULT VOCAL FOLD HISTOLOGY Although the larynx serves the critical evolutionary function of airway protection and material clearance, its highly complex endolaryngeal anatomy and mechanics play a central role in human phonation. The membranous portions of fully developed human vocal folds have a specialized layered structure, vital for producing a predictable, sustained and dynamic www.co-otolaryngology.com
voice. The superficial layer of the vocal fold is nonkeratinizing stratified squamous epithelium capable of sustaining the repetitive trauma of phonation, throat clearing and coughing. At the deepest aspect of the mucosal layer is the basement membrane, important for mucosal adherence and also a histologic barrier to the spread of malignancy. Below the epithelium is the lamina propria, which is subdivided into superficial, intermediate and deep layers. The stratification of the lamina propria is defined by varying histologic features from superficial to deep. Extracellular matrix (ECM) components, secreted by the lamina propria cells or fibroblasts, exist in different quantities throughout the three layers. The superficial lamina propria (SLP), also known as Reinke’s space, is acellular and contains glycoproteins, mucopolysaccharides, water and loosely arranged collagen fibers that yield a viscous,
Department of Otolaryngology – Head and Neck Surgery, University of California, Davis, Sacramento, California, USA Correspondence to Maggie A. Kuhn, MD, Department of Otolaryngology – Head and Neck Surgery, Center for Voice and Swallowing, University of California, Davis, 2521 Stockton Boulevard, Suite 7200, Sacramento, CA 95817, USA. Tel: +1 916 734 2704; fax: +1 916 703 5011; e-mail: [email protected] Curr Opin Otolaryngol Head Neck Surg 2014, 22:460–465 DOI:10.1097/MOO.0000000000000108 Volume 22 Number 6 December 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Histological changes in vocal fold growth and aging Kuhn
KEY POINTS Contemporary investigations have furthered our understanding of vocal fold histology and have generated disagreement regarding vocal fold development through infancy and childhood. Novel applications of existing technology may make it possible to perform in-vivo evaluations of cross-sectional vocal fold anatomy. Thorough knowledge of vocal fold microanatomy has laid the foundation for current emerging therapeutics including decellularized scaffolds, stem cells and novel augmentation techniques.
gelatinous texture. The composition of the SLP provides the viscoelasticity necessary for proper mucosal support and vibration. Progressing deeper, the layers become more dense and fibrous as the amount of fibroblasts increase and the constitution of the ECM changes. Elastin predominates in the intermediate lamina propria (ILP), and the deep lamina propria (DLP) contains tightly arranged collagen. In each layer, the fibers are organized parallel to the free edge of the vocal fold and vocalis myofibrils. Histologic studies have produced more detailed descriptions of the ECM components and their contribution to vocal fold mechanics. Elastin is responsible for much of the vocal fold’s elastic recoil or stretch properties. In the lamina propria, elastin is present in at least three forms: oxytalan, elaunin or mature elastic fibers [1]. Oxytalan and elaunin predominate in the SLP, whereas the ILP and DLP contain mostly mature elastic fibers. Furthermore, a higher concentration of elastin lies within the deeper layers of the lamina propria, especially at its interface with the vocalis muscle contributing to both vocal fold viscoelasticity and stability [2]. Alternatively, collagen is responsible for transmitting tension from the vocalis muscle to the lamina propria and confers durability by protecting from overextension of elastin [2,3]. The adult vocal fold is composed primarily of thin, type III and wavy, thick type I collagens [4]. At the vocal fold core is the thyroarytenoid muscle (vocalis portion), which is one of the paired, intrinsic adductor muscles of the larynx. Recently, de Campos et al. [5] reported findings from a histologic study, which supported their hypothesis that the vocalis muscle has fibers oriented similarly to the tongue, longitudinal and transverse. This configuration allows the vocalis, and associated vocal fold, to move in abundant ways. In 1974, Hirano [6] conceptualized the microanatomy of the vocal fold with his body-cover
theory, which illustrates how the loose superficial mucosal layer is able to vibrate freely over the stiffer, deeper layers. In his description, the cover is composed of the epithelium and SLP. The middle layer, or ligament, is formed by the ILP and DLP. The thyroarytenoid muscle, specifically the vocalis portion, serves as the body. Acceptable voice production is dependent on the vocal fold cover moving freely over the more rigid ligament and body, thereby producing vibration. During the past decade, another cellular component of the lamina propria has been introduced and described. Vocal fold stellate cells are a special class of interstitial cells similar to fibroblasts that are important for ECM metabolism and responsible for maintenance of vocal fold vibroelasticity. Stellate cells are likely somatic stem or progenitor cells that are desmin positive and characteristically contain perinuclear vitamin A droplets similar to other stellate cells [7]. As with other stem cells, stellate cells are closely associated with a niche. The macula flava, which is implicated in vocal fold growth and development, has recently been identified as the niche for stellate cells [8,9 ]. The activity of stellate cells and resultant ECM metabolism is hypothesized to be stimulated by phonation. Supporting this theory is the recent observation that after more than a decade of aphonia, the lamina propria of a deceased 64-year-old stroke victim was significantly atrophic and homogeneous with decreased ECM components [10]. &
JUVENILE VOCAL FOLD HISTOLOGY Because of the paucity of specimens and poor postmortem quality of larynges, studies on ex-vivo fetal, infantile and pediatric vocal folds are limited. Consequently, much more is understood about vocal fold histology and composition in adults than in neonates or children. On the basis of existing histologic evidence, it is widely accepted that the infant vocal fold lacks the layered structure seen in adults. Functionally, this seems plausible, given the decreased demands on neonatal vocal folds (crying) compared to the adult (speech, intonation, pitch variation). Previous reports show that the lamina propria consists of a monolayer at birth and during early childhood [11], develops into a bilayer structure by age 10 and does not become trilayered until adolescence [12]. In a slight departure, Boseley and Hartnick [13] reported that progression of lamina propria from monolayer to bilayer then ultimately to trilayer occurs by age 7. However, they agree that juvenile vocal fold composition and orientation of elastin and collagen does not achieve adult features until adolescence [14]. Following patterns seen in
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adult vocal folds, those of children demonstrate predominance of elastic fibers in the SLP and ILP, whereas collagen deposition increases in DLP [1]. Recent histologic studies of fetal vocal folds have further challenged what was previously accepted, and there is growing controversy surrounding the presence or absence of a vocal ligament in neonates [15]. De Campos et al.’s [16 ] elegant histologic evaluation of the larynx from a 25-week-old fetus lends further clues to the developing vocal fold morphology. He discovered the presence of a vocal ligament as well as thyroarytenoid fibers running in both longitudinal, as has been previously described, and transverse directions. These findings in fetal vocal folds may insinuate the constitution of neonatal vocal fold histology at birth. The reliability of histologic evaluations of vocal folds was recently tested by Infusino et al. [17 ] who evaluated the ability to identify one, two and three layers of lamina propria histopathologically. Using a repository of human adult, human fetal and porcine vocal fold sections, they found that blinded pathologists had an accuracy of 50–75% in identifying the source of the specimens using histologic features. Although some of the discrepancy may be attributed to processing artifacts or lack of knowledge regarding source of the specimen, these results suggest that the traditional vocal fold histologic development paradigm may be incomplete. Juvenile vocal fold maturation results in voice changes from childhood to adulthood that vary in females and males, particularly during puberty. These changes are a normal consequence of overall growth and development, laryngeal descent as well as vocal fold elongation and microanatomy maturation. Vocal folds are androgen and estrogen sensitive, and shifting hormones are responsible for most of the changes experienced during puberty as well as menopause [18,19,20 ]. Puberty is met with vocal fold histologic changes including muscle thickening, variable lubrication and definitive development of the trilayered lamina propria. Immunohistochemistry studies demonstrate that female and male vocal folds express both estrogen and progesterone receptors [20 ]. In women, these hormone receptors are thought to account for improved voice during certain portions of the menstrual cycle. The distribution, pattern and density of estrogen and progesterone receptors in the vocal folds remain an area of active research. Other histologic features of the pubescent larynx also vary from men to women. Elastin in male vocal folds, for example, is more abundant in the cover than in the ligament producing a higher elastic modulus and stiffer ligament than in female vocal folds [21]. Furthermore, elastin in the male lamina &
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propria is wavy with separation and fragmentation whereas female lamina propria contains more compact elastin [22]. CHANGES IN VOCAL FOLD HISTOLOGY WITH AGING Age-related vocal alterations are characterized by a weak, hoarse, strained or low-pitched voice [23,24], and associated stroboscopic findings include vocal fold bowing, decreased amplitude and reduced mucosal wave [23,25]. Such changes result from coexisting medical conditions, deterioration of the cricoarytenoid joint, cartilage ossification, decreased pulmonary function, impaired motor neuron function and intrinsic vocal fold changes [26]. Among the histologic changes seen with vocal fold aging, alterations to the ECM of the lamina propria are the most significant and result in reduced viscoelasticity. The SLP thins, fibroblast activity declines [27] and the vascular supply diminishes [28], resulting in vocal folds that become less elastic and less pliable with increasing age [29]. Hyaluronic acid, a key element of vocal fold ECM, is significantly reduced in elderly vocal folds [30,31]. Collagen content, particularly mature type I fibers, increases especially in the DLP [29,32]. These fibers are disorganized and irregular when compared to vocal folds of younger individuals. Immature type III collagen deposition decreases and is primarily confined to the SLP [27] in which active tissue repair and collagen turnover continues with aging [33]. Elastin presence decreases in the SLP and ILP after 40 years of age, especially in men [12,29]. Furthermore, remaining fibers grow resulting in thicker elastin fibers in geriatric vocal folds [34]. Changes to vocal fold elastin result in homeostatic alterations in the ECM, thereby reducing the elastic properties and vibratory characteristics of the aged vocal fold [35]. Further changes are sex-specific. In older men, the ILP thins while the DLP undergoes additional collagen deposition and thickens. An additional consequence of aging observed in the vocal fold is loss of muscle bulk and resultant vocal fold atrophy [36,37]. Equipped with a thorough understanding of the age-related histologic changes within the vocal folds, investigators are currently evaluating therapeutic agents that have the potential to slow or reduce the consequences of vocal fold aging. Hepatocyte growth factor, which has potent antifibrotic capacity, is a promising candidate that has recently been studied in a rat model [38,39]. Treatment of vocal folds with hepatocyte growth factor has resulted in increased ECM deposition including hyaluronic acid and matrix metalloproteinases as well as decreased collagen type I (mature) deposition. Volume 22 Number 6 December 2014
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Histological changes in vocal fold growth and aging Kuhn
ANALYSIS OF VOCAL FOLD HISTOLOGY Expanding our knowledge of human vocal fold histology is particularly restricted by our limited ability to evaluate microanatomy in vivo. Challenges such as few available human laryngeal specimens and preservation artifacts have driven the development of novel applications for existing technologies and animal models. Optical coherence tomography (OCT) is a noninvasive imaging technique that utilizes near-infrared light to provide highly detailed cross-sectional images. Ophthalmologists first used OCT in the 1990s to evaluate the fine, layered structure of the retina. Recently, investigators have shown that when used in the larynx, OCT has the potential to produce in-vivo images of subepithelial vocal fold architecture to a resolution of 10 mm and depth of 2 mm [40]. To date, studies in ex-vivo vocal folds have demonstrated OCT’s ability to distinguish layers of the vocal fold and distinguish between specimens with different numbers of layers. Furthermore, preliminary in-vivo studies have reported its successful use in cross-sectional imaging of the newborn airway [41] as well as adult vocal folds [42]. This technology is particularly attractive as it produces in-vivo images of vocal fold microanatomy while posing minimal risk to patients allowing for real-time assessment of lesion depth and further investigation into pediatric vocal fold development. Also important for in-vivo study of vocal fold growth and aging has been establishing a valid and reproducible animal model. Recently described, the senescence-accelerated prone mouse model has a 26% shorter lifespan than other murine models because of rapid acquisition of aged characteristics [43]. These extend to the vocal folds, which exhibit increased collagen density and deposition, decreased hyaluronic acid production and decreased fibroblast activity. Access to such an animal model allows for representative investigations into the mechanisms of vocal fold aging and potential development of therapeutic interventions.
VOCAL FOLD HISTOLOGY IN DISEASE AND TREATMENT Commonly, dysphonia results from disturbances of the intricate vocal fold microanatomy. The importance of the specialized layered vocal fold structure in voice production has been well established, as has the abnormal histopathology of many common voice disorders. Expanding knowledge of vocal fold histology in disease states is critical for understanding implications across the lifespan. Our awareness of voice disorders in the age extremes has grown during recent decades and is being met with a more
comprehensive understanding of the vocal fold histology associated with dysphonia. Vocal fold scar remains one of the more challenging vocal fold pathologies to address. The scar results from severely damaged epithelium and loss of or changes to the ECM of the lamina propria. In the scarred state, changes to the normal ECM include increased procollagen, collagen and fibronectin as well as decreased elastin, hyaluronic acid and overall fiber organization [44]. Unfortunately, available therapies for vocal fold scar do not replace or restore original ECM characteristics [45], but our contemporary understanding of the histologic basis for scar is a foundation for promising developments. Regenerative medicine has flourished in recent years and has also been applied to treating vocal fold disorders. For purposes of restoring vocal fold microanatomy, therapies must address both the cellular and ECM components. Using a rat model, Johnson et al. [46] demonstrated acceptable survival when bone marrow-derived mesenchymal stem cells and a synthetic ECM were injected into vocal folds that had been surgically scarred. Encouragingly, the surrounding vocal fold was also found to have increased procollagen, fibronectin and transforming growth factor-b. In a rabbit model, Kim et al. [47] used adipose-derived mesenchymal stem cells and an alginate matrix to address surgically inflicted vocal fold scar. Their results showed increased collagen and growth factor production as well as subjective improvement in viscoelastic properties of the vocal fold. In a recent proof of concept study, Tse and Long [48 ] produced an acellular vocal fold scaffold through detergent decellularization. The scaffold retains ECM components and shares properties of native vocal fold for potential cell implantation and use in the treatment of vocal fold scar. Apart from regenerative medicine techniques, other novel interventions capitalize on our growing knowledge of vocal fold histology. Microendoscopy of Reinke’s space-guided mini-thyrotomy has recently been described and may represent a possible surgical adjunct for treating scar. A pilot study in human cadavers shows appropriate positioning of carboxymethylcellulose in histologic vocal fold sections [49]. This technique has the potential application in individuals with vocal fold scar or for elderly patients who seek treatment for loss of SLP as a consequence of aging. &
CONCLUSION Human vocal folds posses a highly specialized histoanatomy that is dynamic and largely responsible for voice changes experienced though the lifespan. Recent investigations into the histologic
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characteristics of developing and aging vocal folds enhance our growing body of knowledge about the important relationship between vocal microanatomy and function. Certain elements, including the accepted fetal and pediatric vocal fold histology paradigm have been challenged whereas others including vocal fold scar and senescent vocal fold histology have been substantiated. Most critically, there has been progress in the development of novel ways to assess vocal fold microanatomy and potentially intervene when it is disordered. Continued evolution of our investigations is imperative, particularly as pediatric and elderly voice disorders have gained special attention during the past decade. Important in their diagnosis and treatment is an evidence-based, cohesive description of pediatric vocal fold histology and development. Furthermore, most therapies targeting vocal fold disease do little to restore normal microarchitecture, and this initiative deserves priority as available technologies continue to advance. Finally, the refinement and future development of noninvasive evaluative techniques, such as OCT, will catapult our knowledge and therapeutic potential into the next era of voice care. Acknowledgements None. Conflicts of interest The author has no conflicts of interest to declare.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Gray SD, Chan KJ, Turner B. Dissection plane of the human vocal fold lamina propria and elastin fibre concentration. Acta Otolaryngol 2000; 120:87–91. 2. Hahn MS, Kobler JB, Zeitels SM, Langer R. Quantitative and comparative studies of the vocal fold extracellular matrix II: collagen. Ann Otol Rhinol Laryngol 2006; 115:225–232. 3. Hahn MS, Teply BA, Stevens MM, et al. Collagen composite hydrogels for vocal fold lamina propria restoration. Biomaterials 2006; 27:1104–1109. 4. Sivasankar M, Ivanisevic A. Atomic force microscopy investigation of vocal fold collagen. Laryngoscope 2007; 117:1876–1881. 5. de Campos D, do Nascimento PS, Ellwanger JH, et al. Histological organization is similar in human vocal muscle and tongue–a study of muscles and nerves. J Voice 2012; 26:811.e19–811.e26. 6. Hirano M. Morphological structure of the vocal cord as a vibrator and its variations. Folia Phoniatr 1974; 26:89–94. 7. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in the human vocal fold. Acta Otolaryngol 2003; 123:106–110. 8. Sato K, Umeno H, Nakashima T. Functional histology of the macula flava in the human vocal fold–Part 2: Its role in the growth and development of the vocal fold. Folia Phoniatr Logop 2010; 62:263–270. 9. Sato K, Umeno H, Nakashima T. Vocal fold stem cells and their niche in the & human vocal fold. Ann Otol Rhinol Laryngol 2012; 121:798–803. This manuscript adds important information about vocal fold stem cells and their regulation amidst growing enthusiasm for regenerative medicine. 10. Sato K, Umeno H, Ono T, Nakashima T. Histopathologic study of human vocal fold mucosa unphonated over a decade. Acta Otolaryngol 2011; 131:1319– 1325.
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11. Hirano M, Sato K. Histological color atlas of the human larynx. San Diego, California: Singular Publishing Group; 1993. 12. Hirano M, Kurita S, Nakashima T. Growth, development, and aging of human vocalfolds. In: Bless DM, Abbs JH, editors. Vocal fold physiology. San Diego, California: College-Hill; 1983. 13. Boseley ME, Hartnick CJ. Development of the human true vocal fold: depth of cell layers and quantifying cell types within the lamina propria. Ann Otol Rhinol Laryngol 2006; 115:784–788. 14. Hartnick CJ, Rehbar R, Prasad V. Development and maturation of the pediatric human vocal fold lamina propria. Laryngoscope 2005; 115:4–15. 15. Nita LM, Battlehner CN, Ferreira MA, et al. The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study. J Anat 2009; 215:692– 697. 16. de Campos D, Ellwanger JH, da Costa Rosa JP, et al. Morphology of fetal & vocal fold and associated structures. J Voice 2013; 27:5–10. An elegant discription of fetal vocal fold histology, significant for its contribution to an evolving area of study. 17. Infusino SA, Ojha S, Maurer R, et al. The utility of histopathology in identifying & structural differences among layers of the lamina propria. Int J Pediatr Otorhinolaryngol 2013; 77:1651–1654. This straight-forward evaluation of the accuracy of histologic vocal fold evaluation raises important questions about the method we overwhelmingly use for analysis of vocal microarchitecture. 18. Raj A, Gupta B, Chowdhury A, Chadha S. A study of voice changes in various phases of menstrual cycle and in postmenopausal women. J Voice 2010; 24:363–368. 19. Fischer J, Semple S, Fickenscher G, et al. Do women’s voices provide cues of the likelihood of ovulation? The importance of sampling regime. PLoS One 2011; 6:e24490. 20. Brunings JW, Schepens JJ, Peutz-Kootstra CJ, Kross KW. The expression of & estrogen and progesterone receptors in the human larynx. J Voice 2013; 27:376–380. A study establishing the hormonal susceptibility of vocal folds, important for understanding vocal fold development and aging as well as gender differences. 21. Chan RW, Fu M, Young L, Tirunagari N. Relative contributions of collagen and elastin to elasticity of the vocal fold under tension. Ann Biomed Eng 2007; 35:1471–1483. 22. Kahane JC. Histologic structure and properties of the human vocal folds. Ear Nose Throat J 1988; 67:322. 23. Bloch I, Behrman A. Quantitative analysis of videostroboscopic images in presbylarynges. Laryngoscope 2011; 111 (11 Pt 1):2022–2027. 24. Ford C. Voice restoration in presbyphonia. Arch Otolaryngol Head Neck Surg 2004; 130:1117. 25. Wu D, Jiang JJ, Fang R, et al. G protein-coupled estrogen receptor 1 expression in human vocal fold. Laryngoscope 2013; 123:948–951. 26. Pontes P, Yamasaki R, Behlau M. Morphological and functional aspects of the senile larynx. Folia Phoniatr Logop 2006; 58:151–158. 27. Chen X, Thibeault SL. Characteristics of age-related changes in cultured human vocal fold fibroblasts. Laryngoscope 2008; 118:1700–1704. 28. Sato K, Hirano M. Age-related changes of elastic fibers in the superficial layer of the lamina propria of vocal folds. Ann Otol Rhinol Laryngol 1997; 106:44–48. 29. Roberts T, Morton R, Al-Ali S. Microstructure of the vocal fold in elderly humans. Clin Anat 2011; 24:544–551. 30. Butler JE, Hammond TH, Gray SD. Gender-related differences of hyaluronic acid distribution in the human vocal fold. Laryngoscope 2001; 111: 907–911. 31. Ohno T, Hirano S, Rousseau B. Age-associated changes in the expression and deposition of vocal fold collagen and hyaluronan. Ann Otol Rhinol Laryngol 2009; 118:735–741. 32. Hirano M, Sato K, Nakashima T. Fibroblasts in geriatric vocal fold mucosa. Acta Otolaryngol 2000; 120:336–340. 33. Jimenez-Vergara AC, Munoz-Pinto DJ, Becerra-Bayona S, et al. Influence of glycosaminoglycan identity on vocal fold fibroblast behavior. Acta Biomater 2011; 7:3964–3972. 34. Hammond TH, Gray SD, Butler J, et al. Age- and gender-related elastin distribution changes in human vocal folds. Otolaryngol Head Neck Surg 1998; 119:314–322. 35. Moore J, Thibeault S. Insights into the role of elastin in vocal fold health and disease. J Voice 2012; 26:269–275. 36. Sato T, Tauchi H. Age changes in human vocal muscle. Mech Ageing Dev 1982; 18:67–74. 37. Kersing W, Jennekens FG. Age-related changes in human thyroarytenoid muscles: a histological and histochemical study. Eur Arch Otorhinolaryngol 2004; 261:386–392. 38. Suehiro A, Wright H, Rousseau B. Optimal concentration of hepatocyte growth factor for treatment of the aged rat vocal fold. Laryngoscope 2011; 121:1726–1734. 39. Ohno T, Yoo MJ, Swanson ER, et al. Regeneration of aged rat vocal folds using hepatocyte growth factor therapy. Laryngoscope 2009; 119:1424– 1430. 40. Maturo S, Benboujja F, Boudoux C, Hartnick C. Quantitative distinction of unique vocal fold subepithelial architectures using optical coherence tomography. Ann Otol Rhinol Laryngol 2012; 121:754–760.
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Histological changes in vocal fold growth and aging Kuhn 41. Ridgway JM, Su J, Wright R, et al. Optical coherence tomography of the newborn airway. Ann Otol Rhinol Laryngol 2008; 117:327–334. 42. Kaiser ML, Rubinstein M, Vokes DE, et al. Laryngeal epithelial thickness: a comparison between optical coherence tomography and histology. Clin Otolaryngol 2009; 34:460–466. 43. Kolachala VL, Torres-Gonzalez E, Mwangi S, et al. A senescence accelerated mouse model to study aging in the larynx. Otolaryngol Head Neck Surg 2010; 142:879–885. 44. Thibeault SL, Li W, Gray SD, Chen Z. Instability of extracellular matrix gene expression in primary cell culture of fibroblasts from human vocal fold lamina propria and tracheal scar. Ann Otol Rhinol Laryngol 2002; 111: 8–14. 45. Hirano S. Current treatment of vocal fold scarring. Curr Opin Otolaryngol Head Neck Surg 2005; 13:143–147.
46. Johnson BQ, Fox R, Chen X, Thibeault S. Tissue regeneration of the vocal fold using bone marrow mesenchymal stem cells and synthetic extracellular matrix injections in rats. Laryngoscope 2010; 120:537–545. 47. Kim YM, Oh SH, Choi JS, et al. Adipose-derived stem cell-containing hyaluronic acid/alginate hydrogel improves vocal fold wound healing. Laryngoscope 2014; 124:E64–72. 48. Tse JR, Long JL. Microstructure characterization of a decellularized vocal fold & scaffold for laryngeal tissue engineering. Laryngoscope 2014; 124:E326– E331. This study describes an innovative technique within the field of regenerative medicine applied to the larynx. 49. Bartlett RS, Hoffman HT, Dailey SH, et al. Restructuring the vocal fold lamina propria with endoscopic microdissection. Laryngoscope 2013; 123:2780– 2786.
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Histological changes in vocal fold growth and aging Maggie A. Kuhn
Purpose of review Sophisticated descriptions of the highly specialized vocal fold microarchitecture have been available for over three decades, but how this anatomy evolves with growth and aging remains an area of active investigation and, at times, a source of controversy. As our aging population expands and interest in pediatric voice disorders blossoms, it is timely to consider our contemporary understanding of evolving vocal fold histology and its implications for voice production. Recent findings Novel applications of existing and emerging biotechnology, development of animal models and skillful use of human specimens have afforded greater insights into the histologic vocal fold changes seen throughout the lifespan in health and disease. Summary Burgeoning knowledge has laid the foundation for more comprehensive models of vocal fold histology and has led to the development of innovative therapies for challenging voice disorders. Keywords aging, development, extracellular matrix, lamina propria, vocal fold histology
INTRODUCTION The specialized microanatomy of the vocal folds is essential for the complex mechanism of voice production. Intimate knowledge of vocal fold histology is fundamental for understanding voice disorders and necessary for approaching treatment. This review begins with a discussion of the most recent expansion of our knowledge regarding normal adult vocal fold histology. As with much of the human anatomy, the vocal folds’ histologic features transform during development and throughout the lifespan. Some changes are a consequence of normal growth and aging, whereas others represent disease states. Recent contributions to our growing understanding of these changes will be addressed. Lastly, the development of promising, novel therapeutic interventions on the basis of our understanding of vocal fold histophysiology will be reviewed.
NORMAL ADULT VOCAL FOLD HISTOLOGY Although the larynx serves the critical evolutionary function of airway protection and material clearance, its highly complex endolaryngeal anatomy and mechanics play a central role in human phonation. The membranous portions of fully developed human vocal folds have a specialized layered structure, vital for producing a predictable, sustained and dynamic www.co-otolaryngology.com
voice. The superficial layer of the vocal fold is nonkeratinizing stratified squamous epithelium capable of sustaining the repetitive trauma of phonation, throat clearing and coughing. At the deepest aspect of the mucosal layer is the basement membrane, important for mucosal adherence and also a histologic barrier to the spread of malignancy. Below the epithelium is the lamina propria, which is subdivided into superficial, intermediate and deep layers. The stratification of the lamina propria is defined by varying histologic features from superficial to deep. Extracellular matrix (ECM) components, secreted by the lamina propria cells or fibroblasts, exist in different quantities throughout the three layers. The superficial lamina propria (SLP), also known as Reinke’s space, is acellular and contains glycoproteins, mucopolysaccharides, water and loosely arranged collagen fibers that yield a viscous,
Department of Otolaryngology – Head and Neck Surgery, University of California, Davis, Sacramento, California, USA Correspondence to Maggie A. Kuhn, MD, Department of Otolaryngology – Head and Neck Surgery, Center for Voice and Swallowing, University of California, Davis, 2521 Stockton Boulevard, Suite 7200, Sacramento, CA 95817, USA. Tel: +1 916 734 2704; fax: +1 916 703 5011; e-mail: [email protected] Curr Opin Otolaryngol Head Neck Surg 2014, 22:460–465 DOI:10.1097/MOO.0000000000000108 Volume 22 Number 6 December 2014
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Histological changes in vocal fold growth and aging Kuhn
KEY POINTS Contemporary investigations have furthered our understanding of vocal fold histology and have generated disagreement regarding vocal fold development through infancy and childhood. Novel applications of existing technology may make it possible to perform in-vivo evaluations of cross-sectional vocal fold anatomy. Thorough knowledge of vocal fold microanatomy has laid the foundation for current emerging therapeutics including decellularized scaffolds, stem cells and novel augmentation techniques.
gelatinous texture. The composition of the SLP provides the viscoelasticity necessary for proper mucosal support and vibration. Progressing deeper, the layers become more dense and fibrous as the amount of fibroblasts increase and the constitution of the ECM changes. Elastin predominates in the intermediate lamina propria (ILP), and the deep lamina propria (DLP) contains tightly arranged collagen. In each layer, the fibers are organized parallel to the free edge of the vocal fold and vocalis myofibrils. Histologic studies have produced more detailed descriptions of the ECM components and their contribution to vocal fold mechanics. Elastin is responsible for much of the vocal fold’s elastic recoil or stretch properties. In the lamina propria, elastin is present in at least three forms: oxytalan, elaunin or mature elastic fibers [1]. Oxytalan and elaunin predominate in the SLP, whereas the ILP and DLP contain mostly mature elastic fibers. Furthermore, a higher concentration of elastin lies within the deeper layers of the lamina propria, especially at its interface with the vocalis muscle contributing to both vocal fold viscoelasticity and stability [2]. Alternatively, collagen is responsible for transmitting tension from the vocalis muscle to the lamina propria and confers durability by protecting from overextension of elastin [2,3]. The adult vocal fold is composed primarily of thin, type III and wavy, thick type I collagens [4]. At the vocal fold core is the thyroarytenoid muscle (vocalis portion), which is one of the paired, intrinsic adductor muscles of the larynx. Recently, de Campos et al. [5] reported findings from a histologic study, which supported their hypothesis that the vocalis muscle has fibers oriented similarly to the tongue, longitudinal and transverse. This configuration allows the vocalis, and associated vocal fold, to move in abundant ways. In 1974, Hirano [6] conceptualized the microanatomy of the vocal fold with his body-cover
theory, which illustrates how the loose superficial mucosal layer is able to vibrate freely over the stiffer, deeper layers. In his description, the cover is composed of the epithelium and SLP. The middle layer, or ligament, is formed by the ILP and DLP. The thyroarytenoid muscle, specifically the vocalis portion, serves as the body. Acceptable voice production is dependent on the vocal fold cover moving freely over the more rigid ligament and body, thereby producing vibration. During the past decade, another cellular component of the lamina propria has been introduced and described. Vocal fold stellate cells are a special class of interstitial cells similar to fibroblasts that are important for ECM metabolism and responsible for maintenance of vocal fold vibroelasticity. Stellate cells are likely somatic stem or progenitor cells that are desmin positive and characteristically contain perinuclear vitamin A droplets similar to other stellate cells [7]. As with other stem cells, stellate cells are closely associated with a niche. The macula flava, which is implicated in vocal fold growth and development, has recently been identified as the niche for stellate cells [8,9 ]. The activity of stellate cells and resultant ECM metabolism is hypothesized to be stimulated by phonation. Supporting this theory is the recent observation that after more than a decade of aphonia, the lamina propria of a deceased 64-year-old stroke victim was significantly atrophic and homogeneous with decreased ECM components [10]. &
JUVENILE VOCAL FOLD HISTOLOGY Because of the paucity of specimens and poor postmortem quality of larynges, studies on ex-vivo fetal, infantile and pediatric vocal folds are limited. Consequently, much more is understood about vocal fold histology and composition in adults than in neonates or children. On the basis of existing histologic evidence, it is widely accepted that the infant vocal fold lacks the layered structure seen in adults. Functionally, this seems plausible, given the decreased demands on neonatal vocal folds (crying) compared to the adult (speech, intonation, pitch variation). Previous reports show that the lamina propria consists of a monolayer at birth and during early childhood [11], develops into a bilayer structure by age 10 and does not become trilayered until adolescence [12]. In a slight departure, Boseley and Hartnick [13] reported that progression of lamina propria from monolayer to bilayer then ultimately to trilayer occurs by age 7. However, they agree that juvenile vocal fold composition and orientation of elastin and collagen does not achieve adult features until adolescence [14]. Following patterns seen in
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adult vocal folds, those of children demonstrate predominance of elastic fibers in the SLP and ILP, whereas collagen deposition increases in DLP [1]. Recent histologic studies of fetal vocal folds have further challenged what was previously accepted, and there is growing controversy surrounding the presence or absence of a vocal ligament in neonates [15]. De Campos et al.’s [16 ] elegant histologic evaluation of the larynx from a 25-week-old fetus lends further clues to the developing vocal fold morphology. He discovered the presence of a vocal ligament as well as thyroarytenoid fibers running in both longitudinal, as has been previously described, and transverse directions. These findings in fetal vocal folds may insinuate the constitution of neonatal vocal fold histology at birth. The reliability of histologic evaluations of vocal folds was recently tested by Infusino et al. [17 ] who evaluated the ability to identify one, two and three layers of lamina propria histopathologically. Using a repository of human adult, human fetal and porcine vocal fold sections, they found that blinded pathologists had an accuracy of 50–75% in identifying the source of the specimens using histologic features. Although some of the discrepancy may be attributed to processing artifacts or lack of knowledge regarding source of the specimen, these results suggest that the traditional vocal fold histologic development paradigm may be incomplete. Juvenile vocal fold maturation results in voice changes from childhood to adulthood that vary in females and males, particularly during puberty. These changes are a normal consequence of overall growth and development, laryngeal descent as well as vocal fold elongation and microanatomy maturation. Vocal folds are androgen and estrogen sensitive, and shifting hormones are responsible for most of the changes experienced during puberty as well as menopause [18,19,20 ]. Puberty is met with vocal fold histologic changes including muscle thickening, variable lubrication and definitive development of the trilayered lamina propria. Immunohistochemistry studies demonstrate that female and male vocal folds express both estrogen and progesterone receptors [20 ]. In women, these hormone receptors are thought to account for improved voice during certain portions of the menstrual cycle. The distribution, pattern and density of estrogen and progesterone receptors in the vocal folds remain an area of active research. Other histologic features of the pubescent larynx also vary from men to women. Elastin in male vocal folds, for example, is more abundant in the cover than in the ligament producing a higher elastic modulus and stiffer ligament than in female vocal folds [21]. Furthermore, elastin in the male lamina &
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propria is wavy with separation and fragmentation whereas female lamina propria contains more compact elastin [22]. CHANGES IN VOCAL FOLD HISTOLOGY WITH AGING Age-related vocal alterations are characterized by a weak, hoarse, strained or low-pitched voice [23,24], and associated stroboscopic findings include vocal fold bowing, decreased amplitude and reduced mucosal wave [23,25]. Such changes result from coexisting medical conditions, deterioration of the cricoarytenoid joint, cartilage ossification, decreased pulmonary function, impaired motor neuron function and intrinsic vocal fold changes [26]. Among the histologic changes seen with vocal fold aging, alterations to the ECM of the lamina propria are the most significant and result in reduced viscoelasticity. The SLP thins, fibroblast activity declines [27] and the vascular supply diminishes [28], resulting in vocal folds that become less elastic and less pliable with increasing age [29]. Hyaluronic acid, a key element of vocal fold ECM, is significantly reduced in elderly vocal folds [30,31]. Collagen content, particularly mature type I fibers, increases especially in the DLP [29,32]. These fibers are disorganized and irregular when compared to vocal folds of younger individuals. Immature type III collagen deposition decreases and is primarily confined to the SLP [27] in which active tissue repair and collagen turnover continues with aging [33]. Elastin presence decreases in the SLP and ILP after 40 years of age, especially in men [12,29]. Furthermore, remaining fibers grow resulting in thicker elastin fibers in geriatric vocal folds [34]. Changes to vocal fold elastin result in homeostatic alterations in the ECM, thereby reducing the elastic properties and vibratory characteristics of the aged vocal fold [35]. Further changes are sex-specific. In older men, the ILP thins while the DLP undergoes additional collagen deposition and thickens. An additional consequence of aging observed in the vocal fold is loss of muscle bulk and resultant vocal fold atrophy [36,37]. Equipped with a thorough understanding of the age-related histologic changes within the vocal folds, investigators are currently evaluating therapeutic agents that have the potential to slow or reduce the consequences of vocal fold aging. Hepatocyte growth factor, which has potent antifibrotic capacity, is a promising candidate that has recently been studied in a rat model [38,39]. Treatment of vocal folds with hepatocyte growth factor has resulted in increased ECM deposition including hyaluronic acid and matrix metalloproteinases as well as decreased collagen type I (mature) deposition. Volume 22 Number 6 December 2014
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Histological changes in vocal fold growth and aging Kuhn
ANALYSIS OF VOCAL FOLD HISTOLOGY Expanding our knowledge of human vocal fold histology is particularly restricted by our limited ability to evaluate microanatomy in vivo. Challenges such as few available human laryngeal specimens and preservation artifacts have driven the development of novel applications for existing technologies and animal models. Optical coherence tomography (OCT) is a noninvasive imaging technique that utilizes near-infrared light to provide highly detailed cross-sectional images. Ophthalmologists first used OCT in the 1990s to evaluate the fine, layered structure of the retina. Recently, investigators have shown that when used in the larynx, OCT has the potential to produce in-vivo images of subepithelial vocal fold architecture to a resolution of 10 mm and depth of 2 mm [40]. To date, studies in ex-vivo vocal folds have demonstrated OCT’s ability to distinguish layers of the vocal fold and distinguish between specimens with different numbers of layers. Furthermore, preliminary in-vivo studies have reported its successful use in cross-sectional imaging of the newborn airway [41] as well as adult vocal folds [42]. This technology is particularly attractive as it produces in-vivo images of vocal fold microanatomy while posing minimal risk to patients allowing for real-time assessment of lesion depth and further investigation into pediatric vocal fold development. Also important for in-vivo study of vocal fold growth and aging has been establishing a valid and reproducible animal model. Recently described, the senescence-accelerated prone mouse model has a 26% shorter lifespan than other murine models because of rapid acquisition of aged characteristics [43]. These extend to the vocal folds, which exhibit increased collagen density and deposition, decreased hyaluronic acid production and decreased fibroblast activity. Access to such an animal model allows for representative investigations into the mechanisms of vocal fold aging and potential development of therapeutic interventions.
VOCAL FOLD HISTOLOGY IN DISEASE AND TREATMENT Commonly, dysphonia results from disturbances of the intricate vocal fold microanatomy. The importance of the specialized layered vocal fold structure in voice production has been well established, as has the abnormal histopathology of many common voice disorders. Expanding knowledge of vocal fold histology in disease states is critical for understanding implications across the lifespan. Our awareness of voice disorders in the age extremes has grown during recent decades and is being met with a more
comprehensive understanding of the vocal fold histology associated with dysphonia. Vocal fold scar remains one of the more challenging vocal fold pathologies to address. The scar results from severely damaged epithelium and loss of or changes to the ECM of the lamina propria. In the scarred state, changes to the normal ECM include increased procollagen, collagen and fibronectin as well as decreased elastin, hyaluronic acid and overall fiber organization [44]. Unfortunately, available therapies for vocal fold scar do not replace or restore original ECM characteristics [45], but our contemporary understanding of the histologic basis for scar is a foundation for promising developments. Regenerative medicine has flourished in recent years and has also been applied to treating vocal fold disorders. For purposes of restoring vocal fold microanatomy, therapies must address both the cellular and ECM components. Using a rat model, Johnson et al. [46] demonstrated acceptable survival when bone marrow-derived mesenchymal stem cells and a synthetic ECM were injected into vocal folds that had been surgically scarred. Encouragingly, the surrounding vocal fold was also found to have increased procollagen, fibronectin and transforming growth factor-b. In a rabbit model, Kim et al. [47] used adipose-derived mesenchymal stem cells and an alginate matrix to address surgically inflicted vocal fold scar. Their results showed increased collagen and growth factor production as well as subjective improvement in viscoelastic properties of the vocal fold. In a recent proof of concept study, Tse and Long [48 ] produced an acellular vocal fold scaffold through detergent decellularization. The scaffold retains ECM components and shares properties of native vocal fold for potential cell implantation and use in the treatment of vocal fold scar. Apart from regenerative medicine techniques, other novel interventions capitalize on our growing knowledge of vocal fold histology. Microendoscopy of Reinke’s space-guided mini-thyrotomy has recently been described and may represent a possible surgical adjunct for treating scar. A pilot study in human cadavers shows appropriate positioning of carboxymethylcellulose in histologic vocal fold sections [49]. This technique has the potential application in individuals with vocal fold scar or for elderly patients who seek treatment for loss of SLP as a consequence of aging. &
CONCLUSION Human vocal folds posses a highly specialized histoanatomy that is dynamic and largely responsible for voice changes experienced though the lifespan. Recent investigations into the histologic
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characteristics of developing and aging vocal folds enhance our growing body of knowledge about the important relationship between vocal microanatomy and function. Certain elements, including the accepted fetal and pediatric vocal fold histology paradigm have been challenged whereas others including vocal fold scar and senescent vocal fold histology have been substantiated. Most critically, there has been progress in the development of novel ways to assess vocal fold microanatomy and potentially intervene when it is disordered. Continued evolution of our investigations is imperative, particularly as pediatric and elderly voice disorders have gained special attention during the past decade. Important in their diagnosis and treatment is an evidence-based, cohesive description of pediatric vocal fold histology and development. Furthermore, most therapies targeting vocal fold disease do little to restore normal microarchitecture, and this initiative deserves priority as available technologies continue to advance. Finally, the refinement and future development of noninvasive evaluative techniques, such as OCT, will catapult our knowledge and therapeutic potential into the next era of voice care. Acknowledgements None. Conflicts of interest The author has no conflicts of interest to declare.
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