Human Vocal Fold Research Articles

Vascularity in the macula flava of human vocal fold as a stem cell niche

ObjectivesThere is growing evidence that the maculae flavae of the human vocal fold are a stem cell niche, which is a microenvironment nurturing tissue stem cells. This study investigated the microenvironment, especially vascularity, in the maculae flavae of the human vocal fold. MethodsThree normal human adult, three normal newborn vocal folds obtained from autopsy cases and three surgical specimens of glottic carcinoma were investigated using light and electron microscopy. For scanning electron microscopy, a chemical digestion method (modified sodium hydroxide maceration method) was used to observe the inner 3-dimensional structure of the macula flava. ResultsCapillaries ran around the anterior and posterior maculae flavae in adults and newborns. However, there was no vascularity in the maculae flavae of the vocal fold. The inner 3-dimensional electron microscopic structure of the macula flava showed there were no blood vessels in the maculae flavae of the vocal fold. Glottic carcinoma (squamous cell carcinoma) surrounded and was in contact with the macula flava, however, the carcinoma did not invade the macula flava indicating there was no vascular supply into the macula flava from the surrounding tissue. ConclusionsThere was no vascularity in the anterior and posterior maculae flavae in the human adult and newborn vocal folds. The present study is consistent with the hypothesis that the hypoxic microenvironment in the maculae flavae of the adult and newborn vocal fold as a stem cell niche is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.

The life-cycle and restoration of the human vocal fold.

To better understand the challenges of designing therapies to treat damaged vocal fold lamina propria, it is essential to understand the biophysical and pathophysiological mechanisms involved in vocal fold development, maintenance, injury, and aging. This review critically analyses these points to try and direct future efforts and new strategies toward science-based solutions. MEDLINE, Ovid Embase, and Wed of Science databases were used to identify relevant literature. A scoping review was performed following the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews checklist. The layered arrangement of the vocal fold, develops during early childhood and is maintained during adulthood unless injury occurs. The stellate cells of the macular flava are likely to be important in this process. The capacity for vocal fold regeneration and growth is lost during adulthood and repair results in the deposition of fibrous tissue from resident fibroblasts. With advancing age, viscoelastic tissue declines, possibly due to cell senescence. Strategies aimed at replacing fibrous tissue within the vocal folds must either stimulate resident cells or implant new cells to secrete healthy extracellular protein. Injection of basic fibroblast growth factor is the most widely reported therapy that aims to achieve this. The pathways involved in vocal fold development, maintenance and aging are incompletely understood. Improved understanding has the potential to identify new treatment targets that could potentially overcome loss of vocal fold vibratory tissue.

Ultrastructures of Colony-forming Tissue Stem Cells in the Human Newborn Vocal Fold Mucosa in Vivo

ヒト声帯黄斑内の細胞をin vitroで培養すると，幹細胞に特徴的なコロニーを形成して増殖する．新生児生体内の声帯黄斑内にコロニー形成が存在するのか，またその超微形態を検討した．病変がないヒト新生児の声帯3例の声帯黄斑の細胞を透過型電子顕微鏡下に観察した．新生児の声帯黄斑にコロニーを形成する細胞群が存在し，細胞から分泌された無定形物質，糖タンパク質を介して，細胞同士が接近・接着し，細胞が集塊していた．接近した細胞は接着複合体（アドヘレンス結合，デスモゾーム様結合）を形成し，これらを介して，またcellular interdigitationを形成し細胞同士が接触していた．幹細胞はin vitroでコロニーを形成して増殖するが，その機序と役割は不明な点が少なくない．新生児声帯黄斑内の組織幹細胞は，生体内（in vivo）でもコロニーを形成していることが確認された.

Методика исследования вибрационной активности голосовых связок

In order to develop equipment for diagnosing the state of the human vocal folds (VF) and monitoring their activity during treatment, a complex of studies of animal VF samples (rabbits) was performed. A highly sensitive non-contact laser-optical equipment and a technique for recording VF vibrations in an air flow with controlled pressure have been developed. For a group of rabbits of the same age, data are given on the registration of vibrational frequencies in healthy VFs and received as a result of therapeutic effects ("treated" VFs). It was found that when air is passed through the trachea under a slight pressure of 10-20 mm Hg. three peaks of the characteristic frequencies of natural mechanical vibrations of the VF appear in the range of ~ 200 - 1000 Hz. As a rule, this is the fundamental frequency f1 and its two harmonics f2 = 2 f1 and f3 = 3 f1. Best of all, these oscillations are excited at the end of the outflow of air under a pressure of 1 - 5 mm Hg. A difference was found in the excited vibrations at low frequencies in healthy VF and VF with defects in the form of a scar on one ligament. The frequencies of the lowest natural vibrations in a defective VF are somewhat higher compared to the lowest frequency in a healthy VF, which is explained by an increase in its rigidity during the formation of scar tissue. The technique is characterized by ease of registration, sensitivity and informativeness during treatment by methods of regenerative medicine. Diagnosis of the state of the VF during treatment in comparison with the data for a healthy VF seems to be informative and useful for restoring the vibrational properties of the vocal cords during the treatment.

Three-dimensional fine structures of the maculae flavae of the human vocal fold using correlative light and electron microscopy.

To analyze various aspects of complex tissue, there is increasing demand to study each sample at different length scales in biology. Correlative light and electron microscopy (CLEM) is the latest technique to correlate two different types of information on the exact same histological area of interest: histology (from light microscopy) and ultrastructure (from electron microscopy). The three-dimensional fine structures of the maculae flavae (MFe) of the human vocal fold were investigated using CLEM. Five normal human adult vocal folds as specimens embedded in paraffin, sectioned, and mounted on glass slides with/without a chemical digestion method (modified sodium hydroxide maceration method) were investigated. Observations using CLEM were performed. The fine structures of cells and extracellular matrices in the MFe and their peripheral regions were able to be observed on the exact same histological area of interest with the light microscope and field emission-scanning electron microscope. Cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells were intermingled in the MFe of the human vocal fold. The extracellular matrices surrounding each three types of cell in the MFe differed, suggesting the cells were different in functional property. CLEM is a useful technique to observe the three-dimensional fine structures of the human vocal fold mucosa. The results of the present study are consistent with the hypothesis that the cells in the MFe of the human vocal fold have heterogeneity and each three types of cell have different properties.

A xenograft study of human adipose stromal cell-based vocal fold mucosal replacement in rabbits.

ObjectivesVocal fold (VF) scarring, manifested by increased collagen, decreased glycosaminoglycans (GAGs), and disrupted elastic fibers, remains a negative consequence of VF injury or resection. The objective of this study is to compare four reconstructive options after Vf mucosal resection in rabbits. A Cell‐Based Outer Vocal fold Replacement (COVR) using human adipose‐derived mesenchymal stromal cells (hASCs) in fibrin scaffold is directly compared with a decellularized scaffold implant, hASC injection, and resection alone without reconstruction. The primary hypothesis is that the cells‐in‐scaffold construct better reconstitutes the VF structure than either cells or scaffold alone, or than healing by secondary intention.MethodsA total of49 rabbits received bilateral VF cordectomy, followed by either COVR implant, decellularized scaffold implant, hASC injection, or no reconstruction (injured control group). Larynges were harvested after 6 weeks.ResultsHistology demonstrated greater lamina propria thickness, less collagen deposition, and more GAGs in COVR animals versus all other treatment groups. Evidence of persistent human cells was found in about half of the cell‐treated animals. RNA levels of fibrosis pathway and macrophage phenotype markers were statistically unchanged among treatment groups at 6 weeks.ConclusionThese data support the efficacy of COVR implantation in restoring VF microstructure in rabbits. The intact COVR was required; isolated components of decellularized scaffold or injected hASC still produced histologic scarring. We propose that the unique bilayered cell structure within fibrin enables controlled matrix remodeling to minimize wound contraction and fibrosis, and to promote GAG deposition.Level of EvidenceBasic science study

Macrophages alter inflammatory and fibrotic gene expression in human vocal fold fibroblasts

Macrophage phenotypes are simplistically classified as pro-inflammatory (M1) or anti-inflammatory/pro-fibrotic (M2). Phenotypically different macrophages are putatively involved in vocal fold (VF) fibrosis. The current study investigated interactions between macrophages and VF fibroblasts. THP-1 monocyte-derived macrophages were treated with interferon-gamma (IFN-γ), lipopolysaccharide (LPS)/IFN-γ, interleukin-10 (IL10), transforming growth factor-β1 (TGF-β), or interleukin-4 (IL4) for 24 h (M(IFN), M(IFN/LPS), M(IL10), M(TGF), and M(IL4), respectively; M(−) denotes untreated macrophages). Differentially activated macrophages and human VF fibroblasts were co-cultured ± direct contact. Expression of CXCL10, CCN2, ACTA2, FN1, TGM2, and LOX was quantified by real-time polymerase chain reaction. Type I collagen and smooth muscle actin (SMA) were observed by immunofluorescence. CXCL10 and PTGS2 were upregulated in fibroblasts indirectly co-cultured with M(IFN) and M(IFN/LPS). M(TGF) stimulated CCN2, ACTA2, and FN1 in fibroblasts. Enzymes involved in extracellular matrix crosslinking (TGM2, LOX) were increased in monocultured M(IL4) compared to M(−). Direct co-culture with all macrophages increased type I collagen and SMA in fibroblasts. Macrophage phenotypic shift was consistent with stimulation and had downstream differential effects on VF fibroblasts. Direct contact with macrophages, regardless of phenotype, stimulated a pro-fibrotic response in VF fibroblasts. Collectively, these data suggest meaningful interactions between macrophages and fibroblasts mediate fibrosis.

Glycolysis of Tissue Stem Cells in the Macula Flava of Newborn Vocal Fold

There is growing evidence that the cells in the maculae flavae are tissue stem cells and the maculae flavae are a stem cell niche of the human vocal fold mucosa. This study investigated the metabolic activity, especially glycolysis, of the tissue stem cells in the maculae flavae of the human newborn vocal fold. Three normal human newborn vocal folds obtained from autopsy cases were investigated using immunohistochemistry. Among the three phenotypes of cells (cobblestone-like polygonal cells, vocal fold stellate cell-like cells and fibroblast-like spindle cells) in the newborn maculae flavae, a small number of cobblestone-like polygonal cells strongly expressed glucose transporter-1. All three phenotypes of cells in the newborn maculae flavae expressed glycolytic enzymes (hexokinase II, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase A). The cells did not express phosphofructokinase-1 (rate-limiting enzyme of regular glucose metabolism pathway) but sparsely express glucose-6-phosphate dehydrogenase (rate-limiting enzyme) indicating the cells relied more on the pentose phosphate pathway. The cells' expression of lactate dehydrogenase A suggests the maculae flavae of the newborn vocal fold is likely to be an anaerobic microenvironment where cells perform anaerobic glycolysis. The present study is consistent with the hypothesis that the tissue stem cells in the maculae flavae of the newborn vocal fold seem to rely more on anaerobic glycolysis, especially by the pentose phosphate pathway, for energy supply. Already at birth, the metabolism of the tissue stem cells in the maculae flavae of the newborn vocal fold is likely to prevent the formation of toxic reactive oxygen species and is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.

Paracrine Effects of Adipose-Derived Cellular Therapies in an in Vitro Fibrogenesis Model of Human Vocal Fold Scarring

Vocal folds (VF) scarring leads to severe dysphonia which negatively impacts daily life of patients. Current therapeutic options are limited due in large part to the high complexity of the micro-structure of the VF. Innovative therapies derived from adipose tissue such as stromal vascular fraction (SVF) or adipose derived stromal/ stem cells (ASC) are currently being evaluated in this indication and paracrine anti-fibrotic effects are considered as predominant mechanisms. The paracrine anti-fibrotic effects of SVF and ASC from healthy donors were tested in an innovative in vitro fibrogenesis model employing human VF fiboblasts (hVFF) and the principles of macromolecular crowding (MMC). Biosynthesis of collogen and alpha-smooth-muscle actin (αSMA) expression in hVFF were quantified after five days of indirect coculture with ASC or SVF using silver stain, western blot and RT-qPCR analysis. Fibrogenesis was promoted by addition of transforming growth factor beta 1 (TGFβ1) combined with MMC characterized by an enhanced deposition of fibrillar collagens and the acquisition of a myofibroblast phenotype (overexpression of αSMA). Adipose-derived therapies led to a reduction in the αSMA expression and the collagen content was lower in hVFF co-cultivated with SVF. ASC and SVF promoted significant prevention of fibrosis in an in vitro fibrogenesis model through paracrine mechanisms, supporting further development of adipose-derived cellular therapies in VF scarring.

Optimization of Synthetic Vocal Fold Models for Glottal Closure.

Synthetic, self-oscillating models of the human vocal folds are used to study the complex and inter-related flow, structure, and acoustical aspects of voice production. The vocal folds typically collide during each cycle, thereby creating a brief period of glottal closure that has important implications for flow, acoustic, and motion-related outcomes. Many previous synthetic models, however, have been limited by incomplete glottal closure during vibration. In this study, a low-fidelity, two-dimensional, multilayer finite element model of vocal fold flow-induced vibration was coupled with a custom genetic algorithm optimization code to determine geometric and material characteristics that would be expected to yield physiologically-realistic frequency and closed quotient values. The optimization process yielded computational models that vibrated with favorable frequency and closed quotient characteristics. A tradeoff was observed between frequency and closed quotient. A synthetic, self-oscillating vocal fold model with geometric and material properties informed by the simulation outcomes was fabricated and tested for onset pressure, oscillation frequency, and closed quotient. The synthetic model successfully vibrated at a realistic frequency and exhibited a nonzero closed quotient. The methodology described in this study provides potential direction for fabricating synthetic models using isotropic silicone materials that can be designed to vibrate with physiologically-realistic frequencies and closed quotient values. The results also show the potential for a low-fidelity model optimization approach to be used to tune synthetic vocal fold model characteristics for specific vibratory outcomes.

Thermal Effects of CO2, KTP, and Blue Lasers with a Flexible Fiber Delivery System on Vocal Folds

To determine the differences in thermal effects on vocal folds between four fiber-routed lasers. In this experimental laboratory study the thermal effects of an AcuPulse Duo CO2 (CO2 AP), UltraPulse Duo CO2 (CO2 UP), KTP, and Blue laser were analyzed using a Schlieren technique on a human tissue mimicking gel model. Power, laser duration, laser fiber distance to tissue and mode (continuous wave [CW] vs pulsed [P] modes) were evaluated in varying combinations in order to compare the effects of the tested lasers and to explore the individual effect on thermal expansion and incision depth of each setting. The model was validated by comparing the results from the Schlieren model with histology of ex vivo fresh human vocal folds after laser irradiation using a selection of the same laser settings, and calculating the intraclass correlation coefficient (ICC). One thousand ninety-eight Schlieren experiments and 56 vocal cord experiments were conducted. In comparison with CW mode, less thermal expansion occurred in P mode in all lasers, while incisions were deeper in the CO2 and more superficial in the KTP and Blue lasers. The mean thermal expansion was found to be minimally smaller, whereas incision depth was pronouncedly smaller in the KTP and Blue compared to the CO2 lasers. Duration of laser irradiation was the most important factor of influence on thermal expansion and incision depth for all lasers in both CW and P modes. The ICC for consistency between the results of the Schlieren model and the vocal cord histology was classified from fair to excellent, except for the thermal expansion of the Blue laser, which was classified as poor. This study demonstrates important differences in thermal effects between CO2, KTP, and Blue lasers which can be explained by the different physical characteristics of the P modes and divergence of the fiber delivery system. The Schlieren imaging model is a good predictor of the relative thermal effects in vocal fold tissue. Our results can be used as a guidance for ENT surgeons using fiber-routed lasers, in order to achieve effective treatment of vocal fold lesions and prevention of functional impairment of vocal folds.

Role of Colony-Forming Tissue Stem Cells in the Macula Flava of the Human Vocal Fold <i>in Vivo</i>

Role of Colony-Forming Tissue Stem Cells in the Macula Flava of the Human Vocal Fold <i>in Vivo</i>

Subject-Specific Computational Fluid-Structure Interaction Modeling of Rabbit Vocal Fold Vibration.

A full three-dimensional (3D) fluid-structure interaction (FSI) study of subject-specific vocal fold vibration is carried out based on the previously reconstructed vocal fold models of rabbit larynges. Our primary focuses are the vibration characteristics of the vocal fold, the unsteady 3D flow field, and comparison with a recently developed 1D glottal flow model that incorporates machine learning. The 3D FSI model applies strong coupling between the finite-element model for the vocal fold tissue and the incompressible Navier-Stokes equation for the flow. Five different samples of the rabbit larynx, reconstructed from the magnetic resonance imaging (MRI) scans after the in vivo phonation experiments, are used in the FSI simulation. These samples have distinct geometries and a different inlet pressure measured in the experiment. Furthermore, the material properties of the vocal fold tissue were determined previously for each individual sample. The results demonstrate that the vibration and the intraglottal pressure from the 3D flow simulation agree well with those from the 1D flow model based simulation. Further 3D analyses show that the inferior and supraglottal geometries play significant roles in the FSI process. Similarity of the flow pattern with the human vocal fold is discussed. This study supports the effective usage of rabbit larynges to understand human phonation and will help guide our future computational studies that address vocal fold disorders.

Low-strain effective Young’s modulus model and validation for multi-layer vocal fold-based silicone specimens with inclusions

A model of the effective low-strain elastic Young’s modulus of multi-layer stacked composites is proposed, which is capable to account for an arbitrary stacked inclusion. Geometrical and discretization-based model results are validated against measured effective Young’s moduli (from 10 up to 40 kPa) on 14 molded silicone specimens embedding a stiff (298 kPa) inclusion with variable size, position, and stacking. Specimens without inclusion represent the muscle, superficial, and epithelium layers in a human vocal fold with Young’s moduli between 4 and 65 kPa. The proposed model allows to predict the influence of a stiff inclusion, mimicking a structural abnormality or pathology somewhere within the vocal fold, on the low-strain effective Young’s modulus. Quantifying the influence of an inclusion or local stiffening on the vocal fold bio-mechanics is a necessary step toward the understanding and mitigation of structural vocal fold pathologies and associated voice disorders.

Descriptive proteomics of paired human vocal fold and buccal mucosa tissue.

The vast majority of voice disorders is associated with changes of the unique, but delicate, human vocal fold mucosa. The ability to develop new effective treatment methods is significantly limited by the physical inaccessibility and the extremely rare occasions under which healthy tissue biopsies can be obtained. Therefore, the interest in laryngological research has shifted to human oral (buccal) mucosa, a similar and more easily available tissue. The harvesting process is less invasive and accompanied with faster healing and less scarring, compared to vocal fold mucosa. Here we report a descriptive proteomic comparison of paired human buccal and vocal fold mucosa by high‐resolution mass spectrometry (CID‐MS/MS). Our study identified a total of 1575 proteins detected within both tissues that are highly consistent in several crucial biological processes, cellular components, and molecular functions. Hence, our proteomic analysis will provide a fundamental resource for the laryngological research community.

Fine Structures of Colony-forming Tissue Stem Cells in the Macula Flava of the Human Vocal Fold in Vivo

ヒト声帯の黄斑は幹細胞ニッチであり，黄斑に分布する細胞は組織幹細胞である可能性を報告してきた．in vitroで黄斑内の細胞を培養するとコロニーを形成して増殖する．本研究では，声帯黄斑（生体内，in vivo）内のコロニー形成とその微細構造を検討した．

Heterogeneity and Hierarchy of Tissue Stem Cells in the Human Vocal Fold Mucosa

Heterogeneity and Hierarchy of Tissue Stem Cells in the Human Vocal Fold Mucosa

Vortex Formation Times in the Glottal Jet, Measured in a Scaled-Up Model.

In this paper, the timing of vortex formation on the glottal jet is studied using previously published velocity measurements of flow through a scaled-up model of the human vocal folds. The relative timing of the pulsatile glottal jet and the instability vortices are acoustically important since they determine the harmonic and broadband content of the voice signal. Glottis exit jet velocity time series were extracted from time-resolved planar DPIV measurements. These measurements were acquired at four glottal flow speeds (uSS = 16.1–38 cm/s) and four glottis open times (To = 5.67–23.7 s), providing a Reynolds number range Re = 4100–9700 and reduced vibration frequency f* = 0.01–0.06. Exit velocity waveforms showed temporal behavior on two time scales, one that correlates to the period of vibration and another characterized by short, sharp velocity peaks (which correlate to the passage of instability vortices through the glottis exit plane). The vortex formation time, estimated by computing the time difference between subsequent peaks, was shown to be not well-correlated from one vibration cycle to the next. The principal finding is that vortex formation time depends not only on cycle phase, but varies strongly with reduced frequency of vibration. In all cases, a strong high-frequency burst of vortex motion occurs near the end of the cycle, consistent with perceptual studies using synthesized speech.

Sub-millisecond 2D MRI of the vocal fold oscillation using single-point imaging with rapid encoding

ObjectiveThe slow spatial encoding of MRI has precluded its application to rapid physiologic motion in the past. The purpose of this study is to introduce a new fast acquisition method and to demonstrate feasibility of encoding rapid two-dimensional motion of human vocal folds with sub-millisecond resolution.MethodIn our previous work, we achieved high temporal resolution by applying a rapidly switched phase encoding gradient along the direction of motion. In this work, we extend phase encoding to the second image direction by using single-point imaging with rapid encoding (SPIRE) to image the two-dimensional vocal fold oscillation in the coronal view. Image data were gated using electroglottography (EGG) and motion corrected. An iterative reconstruction with a total variation (TV) constraint was used and the sequence was also simulated using a motion phantom.ResultsDynamic images of the vocal folds during phonation at pitches of 150 and 165 Hz were acquired in two volunteers and the periodic motion of the vocal folds at a temporal resolution of about 600 µs was shown. The simulations emphasize the necessity of SPIRE for two-dimensional motion encoding.DiscussionSPIRE is a new MRI method to image rapidly oscillating structures and for the first time provides dynamic images of the vocal folds oscillations in the coronal plane.

Surface kinematic and depth-resolved analysis of human vocal folds in vivo during phonation using optical coherence tomography.

.Significance: The human vocal fold (VF) oscillates in multiple vectors and consists of distinct layers with varying viscoelastic properties that contribute to the mucosal wave. Office-based and operative laryngeal endoscopy are limited to diagnostic evaluation of the VF epithelial surface only and are restricted to axial-plane characterization of the horizontal mucosal wave. As such, understanding of the biomechanics of human VF motion remains limited.Aim: Optical coherence tomography (OCT) is a micrometer-resolution, high-speed endoscopic imaging modality which acquires cross-sectional images of tissue. Our study aimed to leverage OCT technology and develop quantitative methods for analyzing the anatomy and kinematics of in vivo VF motion in the coronal plane.Approach: A custom handheld laryngeal stage was used to capture OCT images with 800 A-lines at 250 Hz. Automated image postprocessing and analytical methods were developed.Results: Novel kinematic analysis of in vivo, long-range OCT imaging of the vibrating VF in awake human subjects is reported. Cross-sectional, coronal-plane panoramic videos of the larynx during phonation are presented with three-dimensional videokymographic and space-time velocity analysis of VF motion.Conclusions: Long-range OCT with automated computational methods allows for cross-sectional dynamic laryngeal imaging and has the potential to broaden our understanding of human VF biomechanics and sound production.