This work was supported by an APART grant of the Austrian Academy of Sciences (CTH), the Technology Agency of the Czech Republic project no. TA04010877 (CTH, VH and JGS), and the Czech Science Foundation (GACR) project no GA16-01246S (to JGS). We thank W. Tecumseh Fitch for his suggestion to use denture fixative cream, and Ing. P. Liska from the Czech Army Forest Service for his help in acquiring the excised deer larynges.

The animal specimens analyzed in this paper were treated in accordance with the standard ethical requirements of the Palacky University in Olomouc, Czech Republic. They stem from red deer living wildly in forests, which were hunted by the Czech Army Forest Service during a regular hunting season.
1. Preparation of the Hemi-larynx Specimen
Note: Only properly prepared specimens should be used, as indicated in4 . Quick freezing of the larynx25 immediately after excision and storage at -80 &#176;C minimizes the potential of tissue degradation and alteration of biomechanical properties, and allows performing the experiments at any convenient time.
<ol>
	<li>Defrosting the larynx
	<ol>
		<li>Insert the frozen larynx into two autoclave bags or any other plastic bags with waterproof sealing. Seal the bags and put them into a water bath heated to 30 &#176;C until the larynx is completely defrosted. The duration required ranges from a few hours to more than a day, depending on larynx size and freezing temperature.</li>
	</ol>
	</li>
	<li>Cleaning the larynx
	<ol>
		<li>After the larynx is defrosted, remove it from the bag and clean it thoroughly with saline solution (0.9% NaCl).</li>
		<li>Carefully remove superfluous tissue as applicable (i.e. external neck muscles, hyoid bone etc.) without damaging the main laryngeal structures, and shorten the trachea to a length adequate for mounting the larynx onto an air supply tube (usually ca. 4-5 cm).</li>
		<li>Check the laryngeal tissue for potential tissue anomalies, such as wounds, organic deformations, or cracks potentially occurring from the freezing process, which could make the larynx unsuitable for the experiment.</li>
	</ol>
	</li>
	<li>Exposure of the thyroid and cricoid cartilages
	<ol>
		<li>Remove parts of the external laryngeal muscle tissue around the thyroid and cricoid cartilage using a scalpel, thus exposing the cartilages in preparation for the mid-sagittal cut creating the hemi-larynx. This preparation stage is depicted in Figure 1A and 1B.</li>
	</ol>
	</li>
	<li>Mid-sagittal cut through the thyroid cartilage
	<ol>
		<li>Make an initial vertical cut through the anterior part of the thyroid cartilage.</li>
		<li>Carefully place the cut slightly more onto the side that is about to be removed, in order not to damage the vocal fold that needs to remain preserved. If possible, use a scalpel for cutting. If the cartilage is ossified, use a small saw.</li>
	</ol>
	</li>
	<li>Cutting the cricoid cartilage
	<ol>
		<li>Lead the cut vertically (inferiorly) from in between the arytenoid cartilages and then through the cricoid cartilage to an approximately horizontal level of the inferior thyroid notch.</li>
	</ol>
	</li>
	<li>Removal of one vocal fold, creating an L-shaped incision in the larynx
	<ol>
		<li>Make a horizontal cut starting from the inferior end of the previously made vertical cut in the cricoid cartilage, and lead the new cut towards the inferior thyroid notch. Anteriorly fold the side of the larynx that is going to be removed.</li>
		<li>Make a vertical cut through the soft tissue on the inner side of the thyroid cartilage - be careful while leading the cut in between the anterior attachment of the vocal folds to the thyroid cartilage, thus avoiding damage to the vocal fold.</li>
	</ol>
	</li>
	<li>Refinement of the cut through the thyroid cartilage
	<ol>
		<li>Use a scalpel, a saw, or a file, in order to apply a precisely straight cut in the thyroid cartilage, and get as close as possible to the anterior part of the previously inspected vocal fold.</li>
		<li>Remove also a small part of the posterior thyroid cartilage, in order to create space for inserting the prong for adducting the arytenoid cartilage and thus the vocal fold (see below). This preparation stage is depicted in Figure 1C and 1D. 
		Note: Depending on the research question, full exposition of the whole vocal fold may be needed to enable its visibility from above. In such a case, the structures above the (true) vocal fold (i.e., the ventricular or vestibular fold, as applicable given the anatomy of the specimen) should be removed. In some specimens the inner soft laryngeal tissue above the vocal folds might lose its connection with the thyroid cartilage and interferes with the vocal fold during vibration, potentially causing spurious (mostly irregular) oscillatory patterns. In such a case careful removal of that tissue is inevitable.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55303/55303fig1.jpg" /> 
Figure 1: Hemi-larynx preparation and mounting. (A) and (B) Cleaned larynx specimen, medial and posterior view, before removal of left vocal fold; (C) and (D) Prepared hemi-larynx with L-shaped incision (left vocal fold removed), medial and posterior view. <a href="//cloudfront.jove.com/files/ftp_upload/55303/55303fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

2. Hemi-larynx Experiment
<ol>
	<li>Hemi-laryngeal setup
	<ol>
		<li>Use an air-supply tube which delivers warmed and humidified air into the larynx.</li>
		<li>Construct two perpendicularly arranged transparent plates as a substitution for removed laryngeal parts.</li>
		<li>Use prongs 4 for increasing the stability of the larynx and creating a proper pre-phonatory larynx configuration by adducting the remaining vocal fold to the vertical glass plate (see Figure 2A). 
		Note: Theoretically, the vocal folds might also be adducted by sutures and weights on a pulley-lever system 26 . However, such an approach has, to the best knowledge of these authors, not yet been attempted for a hemilarynx preparation.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55303/55303fig2.jpg" />
Figure 2: Hemi-larynx setup. (A) Supporting structures: air supply tube, L-shaped glass plate arrangement, adduction prongs. (B) Mounted hemi-larynx preparation with adduction prongs. (C) and (D) Close-ups of hemi-larynx-preparation, viewed from the side and from the top, respectively. <a href="//cloudfront.jove.com/files/ftp_upload/55303/55303fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Mounting the hemi-larynx
	<ol>
		<li>Cover the air supply tube with denture fixative cream and mount the larynx using the remaining part of its trachea. The fixative cream works as an adhesive and closes potential gaps, thus creating an air-tight seal.</li>
		<li>Fasten the trachea with a plastic tightening strap or a hose clamp.&#160;</li>
		<li>Also cover the edges of the cut through the thyroid cartilage with the fixative cream, while avoiding spreading fixative cream on the vocal fold or the inner soft laryngeal tissues.</li>
		<li>Attach the transparent plates.</li>
	</ol>
	</li>
	<li>Stabilization of the thyroid cartilage, adduction of the vocal fold using prongs
	<ol>
		<li>Use the prongs to adduct the vocal fold to the plate and stabilize the thyroid cartilage.</li>
		<li>After the fixative cream has set, apply the airflow in order to establish vocal fold oscillation and check for possible leaks between the hemi-larynx and the glass plates.</li>
		<li>Seal eventually occurring gaps by adding more fixative cream.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The hemi-larynx preparation shares the advantages of the &#34;conventional&#34; (full) excised larynx setup: In such an experimental approach, physical and physiological boundary conditions and parameters (such as subglottal pressure or vocal fold elongation) can be controlled fairly well. The behavior of the hemilarynx is homologous to that of a full larynx with a perfect lateral symmetry, with the exception that magnitudes of some parameters (e.g., air flow rate, sound pressure) are reduced by approximately 50...

Voice is typically created by vibrating laryngeal tissue (mainly the vocal folds), which converts a steady airflow, supplied by the lungs, into a sequence of airflow pulses. The acoustic pressure waveform (i.e., the primary sound) emerging from this sequence of flow pulses acoustically excites the vocal tract which filters them, and the resulting sound is radiated from the mouth and (to a certain degree) from the nose1. The spectral composition of the generated sound is largely influenced by the quality of vocal fold vibration, governed by laryngeal biomechanics and interactions with the tracheal airflow2. Both in a clinical and a research context, documentation and assessment of vocal fold vibration is thus of foremost interest when studying voice production.
In humans, direct endoscopic investigation of the larynx during sound production in vivo is challenging, and it is virtually impossible in nonhuman mammals, given current technological means. Therefore, and in order to guarantee carefully controlled physical and/or physiological experimental boundary conditions, the use of excised larynges3,4 is in many cases an adequate substitution for investigation of in vivo voice production mechanisms.
Vocal fold vibration is a complex three-dimensional phenomenon5. While conventional investigation methods like laryngeal endoscopy (in vivo) or excised larynx preparations typically provide only a superior view of the vibrating vocal folds6, they do not allow for complete three-dimensional analysis of vocal fold motion. In particular, in the superior view the lower (caudal) margins of the vocal folds are invisible during a major portion of the vibratory cycle. This is due to the phase delay between the inferior (caudal) and the superior (cranial) edge of the vocal folds, a phenomenon which is typically seen during vocal fold oscillation5. As direct empirical evidence for backing up findings from mathematical and physical models is scarce, knowledge of the geometry and motion of the lower vocal fold edge7, and thus the geometry of the subglottal channel8,9,10 is crucial for better understanding the interaction between laryngeal airflow, vocal fold tissue, and the resulting forces and pressures11,12. Another aspect of vocal fold vibration that is hidden from the customary superior view is the vertical (caudo-cranial) depth of the contact between the two vocal folds. The vertical contact depth is related to the vertical thickness of the vocal folds, which is a potential indicator of the vocal register used in singing (&#34;chest&#34; vs. &#34;falsetto&#34; register)13,14.
In order to overcome the shortcomings of conventional (full) excised larynx preparations, a so-called hemi-larynx setup can be utilized, where one half of the larynx is removed, thus facilitating the assessment of the vibratory characteristics of the remaining vocal fold in three dimensions. Surprisingly, since the introduction of this setup in the 1960s15 and an initial validation of the concept in 199316, not many laboratories have performed experiments with this promising experimental approach17,18,19,20,21,22,23. An explanation for this might be found in the difficulties of creating a viable hemi-larynx preparation. While the conventional excised (full) larynx preparation is well documented4, no such in-depth instructions are as yet available for creating a hemi-larynx setup. It is therefore the purpose of this paper to provide a tutorial for establishing a reliably reproducible hemi-larynx setup, supplemented by experimental results from red deer specimens.
A hemi-larynx setup shares many features with a &#34;conventional&#34; excised larynx setup, such as measurement equipment, high-speed or other imaging technology to adequately document the vibrations of the laryngeal structures during sound generation, or proper supply of heated, humidified air. These general setup considerations are described in detail in both a book chapter4 and a technical report from the National Center of Voice and Speech24. Reiteration of these instructions would be beyond the scope of this manuscript. Here, only the specialized directives for generating a hemi-larynx setup are presented.

<table><tbody><tr><td>Surgical blades</td><td>Surgeon</td><td>Jai Surgical Ltd., New Delhi, India</td><td></td></tr><tr><td>Saw</td><td>Hand saw (Lux, 150 mm length)</td><td>Lux, Wermelskirchen, Germany</td><td></td></tr><tr><td>Thermometer</td><td>Testo 922</td><td>Testo Ltd., Hampshire, UK</td><td>K-type Probe, Operating temperature -20 to +50 &amp;deg;C</td></tr><tr><td>Autoclave bags</td><td>Autoclave bags</td><td>vwr.com, VWR International s.r.o., Stribrna Skalice, Czech republic</td><td></td></tr><tr><td>Conductive glass plates</td><td>Custom made</td><td>UPOL - Joint laboratory of Optics&lt;br/&gt; Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td><td></td></tr><tr><td>Fixative cream</td><td>Denture fixative cream</td><td>Blend-a-dent Natural</td><td></td></tr><tr><td>Prongs and fastening system</td><td>Customized Kanya Al eloxed profiles</td><td>Distributor: VISIMPEX a.s.. Seifertova 33, 750 02 Prerov, the Czech Rep.;</td><td>&amp;nbsp;Combination of Kanya RVS and PVS fastening systems (http://www.kanya.cz/) + custom made prongs</td></tr><tr><td>Mounting tube</td><td>Custom made</td><td>UPOL - Joint laboratory of Optics,&lt;br/&gt; Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td><td></td></tr><tr><td>LED Light</td><td>Verbatim 52204 LED Lamp</td><td>Mitsubishi Chemical Holdings Corporation, Tokyo, Japan</td><td></td></tr><tr><td>Camera</td><td>Canon EOS1100D</td><td>Canon Inc.</td><td>18-55 mm lens</td></tr><tr><td>Airpump</td><td>Resun LP100</td><td>Resun</td><td></td></tr><tr><td>Strobe light</td><td>ELMED Helio-Strob micro2</td><td>ELMED Dr. Ing. Mense GmbH, Heiligenhaus, Germany</td><td></td></tr><tr><td>Humidifier</td><td>Custom made</td><td>Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td><td></td></tr><tr><td>Subglottic tract</td><td>Custom made adjustable subglottic tract</td><td>Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td><td>Hampala, V., Svec, Jan, Schovanek, P., and Mandat, D. Uzitny vzor c. 25585: Model subglotickeho traktu. [Utility model no. 25585: Model of subglottal tract] (In Czech) Soukup, P. 2013-27834(CZ 25505 U1), 1-7. 24-6-2013. Praha, Urad prumysloveho vlastnictvi</td></tr></tbody></table>

hemi-laryngeal setup for studying vocal fold vibration in three dimensions

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.
Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

Illustrations of the hemi-larynx preparation and its mounting on the air supply tube, as referenced in the previous section, are provided in Figure 1 and Figure 2, respectively.
Documentation of vocal fold vibration from two camera angles
Airflow-induced self-sustaining oscillation of the hemi...

Watch this Scientific Journal Video about Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions at JoVE.com

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

hemi-laryngeal-setup-for-studying-vocal-fold-vibration-three

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.9K Views.  Palacky University Olomouc. This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

Video: Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Bioengineering

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

fMRI Mapping of Brain Activity Associated with the Vocal Production of Consonant and Dissonant Intervals

The neural correlates of consonance and dissonance perception have been widely studied, but not the neural correlates of consonance and dissonance production. The most straightforward manner of musical production is singing, but, from an imaging perspective, it still presents more challenges than listening because it involves motor activity. The accurate singing of musical intervals requires integration between auditory feedback processing and vocal motor control in order to correctly produce each note. This protocol presents a method that permits the monitoring of neural activations associated with the vocal production of consonant and dissonant intervals. Four musical intervals, two consonant and two dissonant, are used as stimuli, both for an auditory discrimination test and a task that involves first listening to and then reproducing given intervals. Participants, all female vocal students at the conservatory level, were studied using functional Magnetic Resonance Imaging (fMRI) during the performance of the singing task, with the listening task serving as a control condition. In this manner, the activity of both the motor and auditory systems was observed, and a measure of vocal accuracy during the singing task was also obtained. Thus, the protocol can also be used to track activations associated with singing different types of intervals or with singing the required notes more accurately. The results indicate that singing dissonant intervals requires greater participation of the neural mechanisms responsible for the integration of external feedback from the auditory and sensorimotor systems than does singing consonant intervals.

The neural correlates of listening to consonant and dissonant intervals have been widely studied, but the neural mechanisms associated with production of consonant and dissonant intervals are less well known. In this article, behavioral tests and fMRI are combined with interval identification and singing tasks to describe these mechanisms.

The neural correlates of consonance and dissonance perception have been widely studied, but not the neural correlates of consonance and dissonance ...

Behavior

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

Medicine

3D Printing of Multilayered Silicone Vocal Fold Models

Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal fold models results in models with undesirable properties, such as stickiness and reproducibility issues. Those vocal fold models are prone to rapid aging, leading to poor comparability across different measurements. In this study, we propose a modification to the manufacturing process by changing the order of layering the silicone material, which leads to the production of non-sticky and highly consistent vocal fold models. We also compare a model produced using this method with a conventionally manufactured vocal fold model that is adversely affected by its sticky surface. We detail the manufacturing process and characterize the properties of the models for potential applications. The outcomes of the study demonstrate the efficacy of the modified fabrication method, highlighting the superior qualities of our non-sticky vocal fold models.&#160;The findings contribute to the development of realistic and reliable vocal fold models for research and clinical applications.

Author Spotlight: Advancements in the Fabrication of Synthetic Vocal Fold Models for Phonetic and Robotic Applications

This study demonstrates the manufacturing of non-sticky and super-soft vocal fold models by introducing a specific way to create the vocal fold layers, providing a detailed description of the manufacturing procedure, and characterizing the properties of the models.

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal ...

Engineering

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Neuroscience

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

Endoscopic Approach for Studying Laryngeal Dysfunction in Rodents

Minimally Invasive Murine Laryngoscopy for Close&#45;Up Imaging of Laryngeal Motion During Breathing and Swallowing

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty&#160;breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions.
To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification).
Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

This protocol describes a serial transoral laryngoscopy approach for mice and rats that permits close-up, unobstructed video imaging of the larynx during breathing and swallowing using an optimized anesthetic regimen and finely tuned endoscopic manipulation techniques.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to ...