Este trabajo fue financiado por una subvención APART de la Academia Austriaca de Ciencias (CTH), la Agencia de tecnología de la República Checa del proyecto no. TA04010877 (CTH, VH y JGS), y la Fundación de la ciencia Checa (GACR) no proyecto 01246S GA16 (a JGS). Agradecemos a W. Tecumseh Fitch su sugerencia de usar crema fijadora de la dentadura y Ing. Liska de P. del servicio de bosque del ejército Checo por su ayuda en la adquisición de las laringes de ciervos suprimido.

El animal muestras analizadas en este trabajo fueron tratadas conforme a los requisitos de la norma éticos de la Universidad Palacky de Olomouc, República Checa. Derivan de vivir violentamente en los bosques, que eran buscados por el ejército Checo bosque servicio durante la temporada regular de la caza de ciervo.
1. preparación de la muestra de Hemi-laringe
Nota: Muestras debidamente preparadas sólo puede usarse, como se indica en4 . Rápida congelación de la laringe25 inmediatamente después de la supresión y almacenamiento a-80 ° C se minimiza el potencial de degradación del tejido y la alteración de las propiedades biomecánicas y permite la realización de los experimentos en cualquier momento conveniente.
<ol><li>
Descongelación de la laringe
	<ol>
<li>Inserte la laringe congeladora en dos bolsas de autoclave o cualquier otras bolsas de plástico con sellado impermeable. Sellar las bolsas y ponerlas en un baño de agua calentado a 30 ° C hasta que la laringe esté completamente descongelada. La duración necesaria oscila entre unas horas y más de un día, dependiendo del tamaño de la laringe y la temperatura de congelación.</li>
	</ol>
</li>
	<li>
Limpieza de la laringe
	<ol>
<li>Después de que la laringe esté descongelada, retire de la bolsa y limpiar con solución salina (0,9% NaCl).</li>
		<li>Retire con cuidado el tejido superfluo como aplicable (músculos del cuello es decir externo, hueso hioides etc.) sin dañar las estructuras laríngeas principal y acortar la tráquea a una longitud adecuada para el montaje de la laringe en un tubo de suministro de aire (generalmente ca. 4-5 cm).</li>
		<li>Compruebe el tejido laríngeo para posibles anomalías del tejido, tales como heridas, deformaciones orgánicas o grietas potencialmente producido por el proceso de congelación, que podría hacer que la laringe no aptos para el experimento.</li>
	</ol>
</li>
	<li>
Exposición de los cartílagos tiroides y cricoides
	<ol>
<li>Saque las piezas del tejido muscular laríngeo externo alrededor del cartílago tiroides y cricoides con un bisturí, exponiendo los cartílagos en preparación para el corte sagital creando la hemi-laringe. Esta etapa de preparación se representa en la figura 1A y 1B.</li>
	</ol>
</li>
	<li>
Corte sagital a través del cartílago tiroides
	<ol>
<li>Hacer un corte vertical inicial a través de la parte anterior del cartílago tiroides.</li>
		<li>Coloque con cuidado el corte un poco más hacia el lado que está a punto de retirarse, para no dañar el doblez vocal que debe permanecer conservados. Si es posible, utilice un bisturí para cortar. Si el cartílago está osificado, use una sierra pequeña.</li>
	</ol>
</li>
	<li>
Cortar cartílago cricoides
	<ol>
<li>Llevar el corte verticalmente (inferiorly) de entre los cartílagos aritenoideo y luego a través del cartílago cricoides a un nivel aproximadamente horizontal de la entalladura de la tiroidea inferior.</li>
	</ol>
</li>
	<li>
Retiro del doblez vocal uno, creando una incisión en forma de L en la laringe
	<ol>
<li>Hacer horizontal cortado a partir del extremo inferior del anterior corte vertical en el cartílago cricoides y conducir el nuevo corte a la muesca de la tiroidea inferior. Pliegue anterior del lado de la laringe que se va a quitar.</li>
		<li>Hacer una vertical corte a través del tejido blando en la cara interna del cartílago tiroides - tenga cuidado mientras que el corte entre el accesorio anterior de las cuerdas vocales hacia el cartílago tiroides, evitando así daños en el pliegue vocal.</li>
	</ol>
</li>
	<li>
Refinamiento del corte a través del cartílago tiroides
	<ol>
<li>Uso de un bisturí, una sierra o un archivo, con el fin de aplicar un corte recto precisamente en el cartílago tiroides y como cerca posible de la parte anterior del doblez vocal previamente inspeccionado.</li>
		<li>Quitar también una parte pequeña del cartílago tiroides posterior, con el fin de crear espacio para la inserción de la clavija para adducting el aritenoides cartílago y, por tanto, el vocal doblan (véase abajo). Esta etapa de preparación se representa en la figura 1C y 1D. Nota: Dependiendo de la pregunta de investigación, exposición completa del doblez vocal entera puede necesitar para permitir su visibilidad desde arriba. En tal caso, deben eliminarse las estructuras por encima del doblez vocal (verdadera) (es decir, la ventricular o vestibular veces, según sea el caso teniendo en cuenta la anatomía del espécimen). En algunos ejemplares el interior suave del tejido laríngeo por encima de las cuerdas vocales puede perder su conexión con el cartílago tiroides e interfiere con el pliegue vocal durante la vibración, potencialmente causando falsos patrones oscilatorios (en su mayoría irregulares). En tal caso es inevitable eliminar de ese tejido.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55303/55303fig1.jpg"> Figura 1 : Montaje y preparación de Hemi-laringe. (A) y (B) limpió laringe espécimen, vista medial y posterior, antes del retiro del doblez vocal izquierdo; (C) y (D) preparado hemi-laringe con incisión en forma de L (a la izquierda del doblez vocal removido), medial y posterior. <a href="//cloudflare.jove.com/files/ftp_upload/55303/55303fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

2. Hemi-laringe experimento
<ol><li>
Configuración de Hemi-laríngea
	<ol>
<li>Utilizar un tubo de suministro de aire que entrega el aire humidificado y calentado en la laringe.</li>
		<li>Construir dos perpendicular había arreglado placas transparentes como una sustitución por piezas desmontadas laríngeas.</li>
		<li>Uso puntas 4 para aumentar la estabilidad de la laringe y la creación de una configuración adecuada de laringe pre-phonatory por adducting del doblez vocal restante a la placa vertical (ver figura 2A). Nota: En teoría, las cuerdas vocales pueden también ser aducción por suturas y pesas en un sistema de polea-palanca 26 . Sin embargo, este enfoque tiene, para el mejor conocimiento de estos autores, aún no han intentado para una preparación hemilarynx.</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55303/55303fig2.jpg">
Figura 2 : Configuración de Hemi-laringe. (A) las estructuras de apoyo: tubo de suministro de aire, arreglo de la placa de vidrio en forma de L dientes de aducción. (B) montaje preparación de hemi-laringe con las horquillas de aducción. (C) y (D) planos de hemi-laringe-preparación, visto desde el lado y desde arriba, respectivamente. <a href="//cloudflare.jove.com/files/ftp_upload/55303/55303fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<ol start="2"><li>
Montaje de la hemi-laringe
	<ol>
<li>Cubrir el tubo de suministro de aire con fijador de prótesis dental crema y montar la laringe con la parte restante de su tráquea. La crema fijadora funciona como adhesivo y cierra brechas posibles, creando un sello hermético.</li>
		<li>Fijar la tráquea con una correa o una abrazadera de apriete de plástico.</li>
		<li>Cubrir también los bordes del corte a través del cartílago tiroides con el crema, evitando separarse crema fijadora en el pliegue vocal o los tejidos blandos de los laringeos interiores fijador.</li>
		<li>Acople las placas transparentes.</li>
	</ol>
</li>
	<li>
Estabilización del cartílago tiroides, aducción del doblez vocal con dientes
	<ol>
<li>Utilice las clavijas para aducción del doblez vocal a la placa y estabilizar el cartílago tiroides.</li>
		<li>Después la crema fijadora, aplicar el flujo de aire con el fin de establecer la oscilación de la cuerda vocal y verifique posibles fugas entre el hemi-laringe y las placas de vidrio.</li>
		<li>Sellar huecos eventualmente ocurrir agregando más crema fijadora.</li>
	</ol>
</li>
</ol>

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<li>Alipour, F., Scherer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+separation+in+a+computational+oscillating+vocal+fold+model%5BTitle%5D)+AND+%22J+Acoust+Soc+Am%22%5BJournal%5D)">Flow separation in a computational oscillating vocal fold model.</a> J Acoust Soc Am. 116 (3), 1710-1719 (2004).</li>
<li>Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+flow+separation+location+on+phonation+onset%5BTitle%5D)+AND+%22J+Acoust+Soc+Am%22%5BJournal%5D)">Influence of flow separation location on phonation onset.</a> J Acoust Soc Am. 124 (3), 1689-1694 (2008).</li>
<li>Kaburagi, T., Tanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-dimensional+models+of+the+glottal+flow+incorporating+viscous-inviscid+interaction%5BTitle%5D)+AND+%22J+Acoust+Soc+Am%22%5BJournal%5D)">Low-dimensional models of the glottal flow incorporating viscous-inviscid interaction.</a> J Acoust Soc Am. 125 (1), 391-404 (2009).</li>
<li>Sidlof, P., Doaré, O., Cadot, O., Chaigne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+flow+separation+in+a+human+vocal+folds+model%5BTitle%5D)+AND+%22Exp+Fluids%22%5BJournal%5D)">Measurement of flow separation in a human vocal folds model.</a> Exp Fluids. 51 (1), 123-136 (2011).</li>
<li>Smith, S. L., Thomson, S. L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356320/">Effect of inferior surface angle on the self-oscillation of a computational vocal fold model.</a> J Acoust Soc Am. 131 (5), 4062-4075 (2012).</li>
<li>Khosla, S., Oren, L., Ying, J., Gutmark, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+simultaneous+measurement+of+intraglottal+geometry+and+velocity+fields+in+excised+larynges%5BTitle%5D)+AND+%22Laryngoscope%22%5BJournal%5D)">Direct simultaneous measurement of intraglottal geometry and velocity fields in excised larynges.</a> Laryngoscope. 124, S1-S13 (2014).</li>
<li>Brücker, C., Kirmse, C., Triep, M. <a target="_blank" href="http://www.ingentaconnect.com/contentone/dav/aaua/2016/00000102/00000002/art00007">Feedback of the glottal jet flow with supraglottal wall oscillations.</a> Acta Acustica United With Acustica. 102 (2), 240-243 (2016).</li>
<li>Herbst, C. T., Fitch, W. T., Lohscheller, J., Svec, J. G. <a target="_blank" href="NULL">AQL 2013, Proceedings of the 10th International Conference on Advances in Quantitative Laryngology, Voice and Speech Research</a>. Deliyski, D. D. , AQL Press. Cincinnati, Ohio, USA. 75-76 (2013).</li>
<li>Berke, G. S., Gerratt, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laryngeal+biomechanics%3A+an+overview+of+mucosal+wave+mechanics%5BTitle%5D)+AND+%22J+Voice%22%5BJournal%5D)">Laryngeal biomechanics: an overview of mucosal wave mechanics.</a> J Voice. 7 (2), 123-128 (1993).</li>
<li>Boessenecker, A., Berry, D. A., Lohscheller, J., Eysholdt, U., Doellinger, M. <a target="_blank" href="http://www.ingentaconnect.com/contentone/dav/aaua/2007/00000093/00000005/art00015">Mucosal wave properties of a human vocal fold.</a> Acta Acustica United With Acustica. 93 (5), 815-823 (2007).</li>
<li>Hirano, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHirano%2C+M+%22Clinical+examination+of+voice%22">Clinical examination of voice</a>. 5, Springer-Verlag. Wien, Austria. (1981).</li>
<li>Jing, B., Tang, S., Wu, L., Wang, S., Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+the+Vibration+of+Laryngeal+Tissue+during+Phonation+Using+Ultrafast+Plane+Wave+Ultrasonography%5BTitle%5D)+AND+%22Ultrasound+in+Med+BIol%22%5BJournal%5D)">Visualizing the Vibration of Laryngeal Tissue during Phonation Using Ultrafast Plane Wave Ultrasonography.</a> Ultrasound in Med BIol. 42 (12), 2812-2825 (2016).</li>
<li>Herbst, C. T. Ch. 6. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHerbst%2C+CT+%22Vertebrate+Sound+Production+and+Acoustic+Communication%22">Vertebrate Sound Production and Acoustic Communication</a>. Suthers, R. A., Fitch, W. T., Fay, R. R., Popper, A. N. , Springer International Publishing. Switzerland. 159-189 (2016).</li>
</ol>

Los autores no tienen nada que revelar.

La preparación de la hemi-laringe comparte las ventajas de la configuración "convencional" laringe extirpada (completo): en este enfoque experimental, condiciones físicas y fisiológicas y parámetros (tales como presión subglótico o alargamiento de cuerdas vocales) pueden ser controlar bastante bien. El comportamiento del hemilarynx es homólogo de una laringe completo con un perfecto lateral simetría, con la excepción de que magnitudes de algunos parámetros (e.g., caudal de aire, presión sonora) se re...

Voz se crea típicamente por vibración del tejido laríngeo (principalmente las cuerdas vocales), que convierte un flujo de aire constante, suministrada por los pulmones, en una secuencia de pulsos de flujo de aire. La forma de onda de presión acústica (es decir, el sonido principal) salen de esta secuencia de pulsos de flujo acústico excita el tracto vocal que filtra, y el sonido resultante es irradiado de la boca y (hasta cierto punto) de la nariz1 . La composición espectral del sonido generado en gran parte está influenciada por la calidad de la vibración vocal, gobernada por la biomecánica laríngea y las interacciones con el flujo de aire traqueal2. Tanto en una clínica y un contexto de investigación, documentación y evaluación de la vibración de la cuerda vocal está así de interés principal al estudiar la producción de la voz.
En los seres humanos, investigación endoscópica directa de la laringe durante la producción de sonido en vivo es un desafío, y es prácticamente imposible en los mamíferos no humanos, medios tecnológicos actuales. Por lo tanto y en orden a la garantía cuidadosamente controlado física o fisiológica experimental condiciones de contorno, el uso de laringes suprimido3,4 es en muchos casos una substitución adecuada para la investigación de en vivo mecanismos de producción de la voz.
Vibración de la cuerda vocal es un fenómeno tridimensional complejo5. Mientras que los métodos de investigación convencionales como endoscopia laríngea (en vivo) o preparaciones de laringe suprimido normalmente proporcionan sólo una vista superior de los pliegues vocales vibrante6, no permiten análisis tridimensional completo de movimiento de la cuerda vocal. En particular, en la vista superior los márgenes inferiores (caudales) de las cuerdas vocales son invisibles durante una parte importante del ciclo vibratorio. Esto es debido al retraso de fase entre el inferior (caudal) y el borde superior (craneal) de las cuerdas vocales, un fenómeno que se ve típicamente durante el doblez vocal oscilación5. Como escasa evidencia empírica directa para respaldar los resultados de modelos matemáticos y físicos, conocimiento de la geometría y el movimiento del vocal baja doble borde7y, por tanto, la geometría del canal subglótico8,9 , 10 es crucial para entender mejor la interacción entre el flujo de aire laríngea, tejido del pliegue vocal y la resultante fuerzas y presiones11,12. Otro aspecto de la vibración de cuerdas vocales que se esconde de la vista superior habitual es la vertical (caudo-craneal) profundidad de contacto entre las dos cuerdas vocales. La profundidad vertical del contacto se relaciona con el espesor vertical de las cuerdas vocales, que es un indicador potencial del registro vocal en canto ("pecho" vs "falsete" registro)13,14.
Para superar las deficiencias de los preparados convencionales laringe extirpada (completo), se puede utilizar una configuración llamada hemi-laringe, donde la mitad de la laringe se quita, lo que facilita la evaluación de las características vibratorias de los restantes del doblez vocal en tres dimensiones. Sorprendentemente, desde la introducción de esta configuración en la década de 196015 y una validación inicial del concepto en 199316, no muchos laboratorios han realizado experimentos con este prometedor enfoque experimental17,18 ,19,20,21,22,23. Una explicación para esto podría encontrarse en las dificultades de crear una preparación viable hemi-laringe. Mientras que la preparación convencional extirpada laringe (completo) está bien documentado4, no hay tales instrucciones más detalladas aún están disponibles para crear una configuración de hemi-laringe. Por lo tanto es el objetivo del presente trabajo proporcionar un tutorial para crear una configuración fiable reproducible hemi-laringe, complementada por los resultados experimentales de ejemplares de ciervo rojo.
Una configuración de hemi-laringe comparte muchas características con una configuración "convencional" laringe suprimido, como equipo de medida, de alta velocidad u otra tecnología de imagen para documentar adecuadamente las vibraciones de las estructuras laríngeas durante la generación de sonido, o correcto suministro de aire calentado y humidificado. Estas consideraciones de configuración general se describen en detalle en un capítulo del libro4 y un informe técnico del Centro Nacional de la voz y el habla24. Reiteración de estas instrucciones sería más allá del alcance de este manuscrito. Aquí, se presentan sólo las directivas especializadas para generar una configuración de hemi-laringe.

<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

La voz de los seres humanos y la mayoría de los mamíferos no humanos se genera en la laringe a través de la oscilación autosostenible de las cuerdas vocales. Documentación visual directa de la vibración de la cuerda vocal es un reto, especialmente en los mamíferos no humanos. Como alternativa, extirpada laringe experimentos proporcionan la oportunidad para investigar la vibración vocal bajo condiciones fisiológicas y físicas controladas. Sin embargo, el uso de una laringe completo simplemente proporciona una vista superior de las cuerdas vocales, excepto las porciones cruciales de las estructuras oscilantes de la observación durante su interacción con las fuerzas aerodinámicas. Esta limitación puede ser superada mediante la utilización de una configuración de hemi-laringe donde la mitad de la laringe se mid-sagittally retira, proporcionando una superior y una vista lateral del doblez vocal restante durante oscilación autosostenida.
Aquí, se da una guía paso a paso para la preparación anatómica de las estructuras de la hemi-laríngea y su montaje en el Banco de laboratorio. Fonación ejemplar de la preparación de la hemi-laringe está documentado con datos de alta velocidad videos capturados por dos cámaras sincronizadas (vistas superiores y lateral), mostrando del movimiento tridimensional del doblez vocal y área contacto variables en el tiempo correspondiente. La documentación de la configuración de la hemi-laringe en esta publicación facilitará la aplicación y constancia fiable en la investigación experimental, los científicos de la voz proporciona el potencial para entender mejor la biomecánica de la producción de la voz.

Ilustraciones de la preparación de la hemi-laringe y su montaje en el tubo de suministro de aire, como se indica en el apartado anterior, se encuentran en la figura 1 y figura 2, respectivamente.
Documentación de la vibración de la cuerda vocal desde dos ángulos de cámara
Inducida por el ...

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

Este papel presenta un protocolo para la preparación de las muestras de hemi-laringe facilitando una visión multidimensional de la vibración de la cuerda vocal, con el fin de investigar diversos aspectos biofísicos de la producción de la voz en los seres humanos y no humanos mamíferos.

Configuración de Hemi-laríngea para el estudio de vibración de la cuerda vocal en tres dimensiones

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

Research

JoVE Journal

Biology

10.9K vistas.  Palacky University Olomouc. Este papel presenta un protocolo para la preparación de las muestras de hemi-laringe facilitando una visión multidimensional de la vibración de la cuerda vocal, con el fin de investigar diversos aspectos biofísicos de la producción de la voz en los seres humanos y no humanos mamíferos.

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.

Sintético, multi-capa, auto-oscilante Fabricación Vocal Modelo Doble

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 ...

Investigación

Bioingeniería

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.

Basada en el chip tridimensional de cultivo celular en perfundido Micro-biorreactores

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

Biología

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.

Investigación De La Separación De Flujo Tridimensional Inducida Por Un Pólipo Modelo De Pliegue Vocal

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.

Construcción y caracterización de un Fold biorreactor Novela Vocal

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

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Técnicas de microinyección para el estudio de la mitosis en el Drosophila melanogaster Embrión sincitial

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Impresión 3D de modelos de cuerdas vocales de silicona multicapa

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.

Autor destacado: Avances en la fabricación de modelos de cuerdas vocales sintéticas para aplicaciones fonéticas y robóticas

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.

Proceso de fabricación para modelos de cuerdas vocales supersuaves no adhesivas

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

Ingeniería

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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.

Preparación de la cuerda vocal de la rata para análisis neuromusculares

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

Configuración de Hemi-laríngea para el estudio de vibración de la cuerda vocal en tres dimensiones

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Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

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Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados en Xenopus Ovocitos