Name: manufacturing process for non-adhesive super-soft vocal fold models
Uploaded: 2024-01-05T14:50:00.000+00:00
Duration: 6 min 24 s
Description: Watch this Scientific Journal Video about Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models at JoVE.com

This project has been supported by the German Research Foundation (DFG), grant no. BI 1639/9-1.

1. Design of the vocal fold models and 3D printing of parts
<ol>
	<li>Create a multi-layered representation of the common M5 geometry of silicone vocal folds using various soft silicone materials. Design the individual parts using computer-aided design (CAD) software. Check the Supplementary Coding File 1, Supplementary Coding File 2, Supplementary Coding File 3, Supplementary Coding File 4, Supplementary Coding File 5, Supplementary Coding File 6, Supplementary Coding File 7, Supplementary Coding File 8 for details. The files are named according to their function in the model and serve as the foundation for the subsequent steps.</li>
	<li>Compile and organize the necessary files for each step in step 2. Refer to the list of required parts and their quantities in Supplementary Figure 1. See a schematic depiction of mold assembly in Supplementary Figure 2.</li>
	<li>Load the STL files into a 3D printing program to generate G-code files that can be read by the 3D printer.</li>
	<li>Prepare the materials (see Table of Materials) for the 3D print.
	<ol>
		<li>For Supplementary Coding File 2 and Supplementary Coding File 5, use a material that causes less visible layer lines, such as polylactic acid (PLA+) or PC.</li>
		<li>For Supplementary Coding File 1, use a harder material such as Tough PLA or Polyethylene terephthalate glycol (PETG) due to its susceptibility to bending stresses. No further restrictions on the choice of printing material apply to the remaining parts.</li>
	</ol>
	</li>
	<li>Adjust the settings of the 3D printing software for the corresponding selected 3D printer.
	<ol>
		<li>For Supplementary Coding File 2 and Supplementary Coding File 5, set a maximum layer height of 0.1 mm.</li>
		<li>For Supplementary Coding File 1, set the infill value to 100% and the print pattern to ZigZag to achieve better stability. Also, set the build plate adhesion category to Skirt instead of Brim, because the geometry of the parts would make it considerably more difficult to remove the brim.</li>
		<li>For the other parts, use the default settings and layer heights of 0.2 mm.</li>
	</ol>
	</li>
	<li>Print the mentioned parts on the 3D printer. Clean the parts and remove any excess material such as brim or printing errors. Smooth the inner contact surfaces with sandpaper (equal or finer than P1000 recommended).</li>
</ol>
2. Fabrication of the vocal fold models
<ol>
	<li>Gather the following parts and materials for creating the body layer: vocal-fold-positiv (2x), vocalis_mold-cap, vocalis_mold-main-part, vocalis_mold-hull, primary silicone, release agent, and thinner (See Table of Materials for details).
	<ol>
		<li>Apply some release agent to the inside surfaces of all the mold parts.</li>
		<li>Assemble the main part and cap of the mold over the positive and place the mold package in the designated pot. Correct the alignment of the two mold parts if necessary. Ensure the hole in the positive for pouring the silicone faces upwards, and the mold has a stable footing on a flat surface.</li>
		<li>Create a mixture of the primary silicone with three parts of thinner (1:1:3), begin by combining component A with the thinner, and subsequently adding component B. Thoroughly mix the components. A total amount of 6 g of silicone mixture is sufficient for casting the body layer from two vocal fold halves.</li>
		<li>Vacuum the silicone mixture in a vacuum chamber at a minimum of -1 bar sub pressure to prevent air bubbles from forming in the cured silicone body.</li>
		<li>Carefully pour the vacuumed silicone mixture into the mold cavity until it appears filled. Fill the surrounding areas of the mold pot to prevent the very thin silicone mixture from sinking through the mold joints. Check the silicone level during the dripping time and add more if necessary. The drip time for this mixture is between 1-2 h.</li>
		<li>After a curing time of about 1 day, but at least 8 h, remove the mold, including the positive, from the pot. Remove the silicone between the mold and the pot before opening the mold.</li>
		<li>When opening the mold, first carefully remove the lid starting from the back of the positive. Then, remove the main body of the mold. Carefully remove any excess silicone using a scalpel or a side cutter.</li>
	</ol>
	</li>
	<li>Prepare the musosa_mold-back, musosa_mold-main-part, and musosa_mold-hull parts, as well as the secondary silicone and release agent for the production of the epithelium layer. (See Table of Materials). 
	NOTE: Steps 2.1 and 2.2 (body and epithelium layer) can be completed simultaneously.
	<ol>
		<li>Assemble the two mold parts and place them in the hull. Prepare the inside of the mold with some release agent, ensuring that the inner walls are coated according to the usage instructions of the respective release agent. Allow the component to air-dry briefly before proceeding.</li>
		<li>Mix a batch of the secondary silicone without using thinner (1:1:0). If air bubbles have been introduced into the silicone mixture while mixing, degas the mixture as in step 2.1.4. 
		NOTE: Be aware of the short drip time of this mixture, which is about 15 min.</li>
		<li>Pour some of the mixture into the mold and swirl it around (leaving the mold in the hull) until all interior surfaces are coated with silicone.</li>
		<li>Turn the mold over and let excess silicone drain out. Secure the mold in this position over a mesh, grate, or at an angle that allows further silicone drainage.</li>
		<li>Prevent the formation of overhangs in the silicone during the curing process by regularly smoothing it out, especially in the area where the air channel will be located. 
		NOTE: These can also be carefully removed later with pliers.</li>
	</ol>
	</li>
	<li>Prepare for the production of the mucosa intermediate layer by preparing the positive with the vocalis silicone layer from step 2.1, the mold prepared with the epithelium layer from step 2.2, and the silicone and thinner as listed in Table of Materials.
	<ol>
		<li>Create a mixture of the primary silicone with five parts of thinner (1:1:5), begin by combining component A with the thinner, and subsequently adding component B. Thoroughly mix the components. A total amount of 4 g of silicone mixture is sufficient.</li>
		<li>Vacuum the silicone mixture in a vacuum chamber as in step 2.1.4.</li>
		<li>Fill a portion of the silicone mixture into the mucosa mold with the prepared epithelial silicone. Tilt the mold until all interior surfaces of the epithelial silicone are covered with a thin layer of oil to facilitate insertion of the positive. 
		NOTE: Optional: Due to the high proportion of diluent, the mixture has a long drip time of several hours during which the mixture can shrink through evaporation. Therefore, wait about 2-3 h before proceeding with the next steps.</li>
		<li>Carefully insert the positive with the vocal body into the mold. Secure the positive in the mold, for example with a clamp, if the positive floats up on the silicone. Depending on the amount of silicone previously added, it may escape at the filling points.</li>
		<li>Fill the mold in the same way as for the casting of the vocalis layer and top up accordingly if the material sinks.</li>
		<li>Wait 24 h after the drip time has ended for the silicone to completely cure.</li>
		<li>After 24 h, remove the body from the mold. First, remove the mold from the shell. Then, starting with the rear section, open the mold and remove the main part of the mold as well.</li>
		<li>Carefully remove any excess silicone, wash the surface and let the body dry.</li>
	</ol>
	</li>
	<li>Mount the two vocal fold halves at the designated locations on the measurement and assembly module in Supplementary Coding File 8. The connection was designed for two M3 screws and M3 square nuts (DIN 562), but they are not mandatory.</li>
</ol>

3D Printing of Multilayered Silicone Vocal Fold Models

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	<li>Drechsel, J. S., Thomson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+supraglottal+structures+on+the+glottal+jet+exiting+a+two-layer+synthetic,+self-oscillating+vocal+fold+model.">Influence of supraglottal structures on the glottal jet exiting a two-layer synthetic, self-oscillating vocal fold model.</a> J Acoust Soc Am. 123 (6), 4434-4445 (2008).</li><li>Stevens, K. A., Shimamura, R., Imagawa, H., Sakakibara, K. I., Tokuda, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validating+Stereo-endoscopy+with+a+synthetic+vocal+fold+model.">Validating Stereo-endoscopy with a synthetic vocal fold model.</a> Acta Acustica United with Acustica. 102 (4), 745-751 (2016).</li><li>Murray, P. R., Thomson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic,+multi-layer,+self-oscillating+vocal+fold+model+fabrication.">Synthetic, multi-layer, self-oscillating vocal fold model fabrication.</a> J Vis Exp. (58), e3498 (2011).</li><li>Spencer, M., Siegmund, T., Mongeau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+superior+surface+strains+and+stresses,+and+vocal+fold+contact+pressure+in+a+synthetic+larynx+model+using+digital+image+correlation.">Determination of superior surface strains and stresses, and vocal fold contact pressure in a synthetic larynx model using digital image correlation.</a> J Acoust Soc Am. 123 (2), 1089-1103 (2008).</li><li>Scherer, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraglottal+pressure+profiles+for+a+symmetric+and+oblique+glottis+with+a+divergence+angle+of+10+degrees.">Intraglottal pressure profiles for a symmetric and oblique glottis with a divergence angle of 10 degrees.</a> J Acoust Soc Am. 109 (4), 1616-1630 (2001).</li><li>Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+human+voice+production+and+control.">Mechanics of human voice production and control.</a> J Acoust Soc Am. 140 (4), 2614-2635 (2016).</li><li>Birkholz, P., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studientexte zur Sprachkommunikation: Elektronische Sprachsignalverarbeitung. , 58-66 (2017).</li><li>Murray, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow-induced responses of normal, bowed, and augmented synthetic vocal fold models. , (2011).</li><li>Alipour, F., Vigmostad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+vocal+folds+elastic+properties+for+continuum+modeling.">Measurement of vocal folds elastic properties for continuum modeling.</a> J Voice. 26 (6), e21-29 (2012).</li><li>Chhetri, D. K., Zhang, Z., Neubauer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+young's+modulus+of+vocal+folds+by+indentation.">Measurement of young's modulus of vocal folds by indentation.</a> J Voice. 25 (1), 1-7 (2011).</li><li>H&#228;sner, P., Birkholz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+aging+of+different+silicone+vocal+folds+models.">Reproducibility and aging of different silicone vocal folds models.</a> J Voice. , (2023).</li><li>Gabriel, F., H&#228;sner, P., Dohmen, E., Borin, D., Birkholz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studientexte zur Sprachkommunikation: Elektronische Sprachsignalverarbeitung. , 221-230 (2019).</li><li>H&#228;sner, P., Prescher, A., Birkholz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+wavy+trachea+walls+on+the+oscillation+onset+pressure+of+silicone+vocal+folds.">Effect of wavy trachea walls on the oscillation onset pressure of silicone vocal folds.</a> J Acoust Soc Am. 149 (1), 466-475 (2021).</li><li>Birkholz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studientexte zur Sprachkommunikation: Elektronische Sprachsignalverarbeitung. , (2016).</li><li>Boersma, P., Weenink, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Praat,+a+system+for+doing+phonetics+by+computer.">Praat, a system for doing phonetics by computer.</a> Glot. Int. 5, 341-345 (2001).</li><li>Fukui, K., Shintaku, E., Honda, M., Takanishi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+vocal+cord+model+for+anthropomorphic+talking+robot+based+on+human+biomechanical+structure.">Mechanical vocal cord model for anthropomorphic talking robot based on human biomechanical structure.</a> Trans Japan Soc Mech Eng Ser C. 73 (734), 2750-2756 (2007).</li><li>Syndergaard, K. L., Dushku, S., Thomson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+conductive+synthetic+vocal+fold+replicas+for+voice+production+research.">Electrically conductive synthetic vocal fold replicas for voice production research.</a> J Acoust Soc Am. 142 (1), 63 (2017).</li></ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The manufacturing process presented here involves critical steps that significantly impact its success. Firstly, it should be noted that the presented manufacturing process does not solve the problem of oil saturation in the vocal fold body material but rather circumvents certain negative side effects. The outgassing and the associated shrinkage and surface waviness still persist, albeit to a lesser extent. A solution to these problems would involve the use of an ultra-soft silicone or alternative material that combines the elasticity modulus of real vocal folds with a stable and durable polymer structure. However, the absence of such a material underscores the ongoing limitations in achieving a comprehensive resolution to these issues. 
The manufacturing process is somewhat more complex than conventional manufacturing methods for vocal fold models consisting of two halves, as it involves more components, and the usual inside-out assembly approach is not applicable here. Inherent advantages include the integrated overflow protection, which facilitates working with highly diluted silicone, and the ability to better observe and react to the filling level and potential bubble formation during the curing process. This is helpful when aiming to minimize manufacturing-induced variations in the properties of models in a small series produced with the same silicone mixtures. Furthermore, it reduces the reject rate.
In comparison to conventional vocal fold modeling, the presented technique offers distinct advantages. With video recordings of the glottal area during oscillation, it was demonstrated that the stickiness of the vocal fold surface could be reduced. As a result, stable flow-induced oscillations could be generated, and clean, artifact-free waveform data could be obtained from the images without the need for aids such as talcum powder or washes before measurement. While the presented conventional model (as the reference) is an extreme example, stickiness is nonetheless an issue for measurements and a risk to the fragile thin epithelium layer. The presented engineering solution can circumvent this problem and contributes to more reliable and reproducible results. 
Looking forward, the modified manufacturing process holds promise for diverse applications. The technique's suitability for producing humanoid robots or speech apparatuses with human-like vocal tracts16 opens avenues for advancements in artificial intelligence and robotics. Furthermore, its application in fundamental research on speech generation and voice production6,17 signifies its potential contribution to the broader scientific community.

Vocal fold models are used to simulate and investigate human voice production under normal and pathological conditions1,2. One of the biggest challenges in creating vocal fold models is to achieve a realistic softness and flexibility that closely approximates those of humans. To achieve these properties, silicone elastomers are often used, which are diluted with high amounts of silicone oil to achieve the corresponding elasticity moduli3,4. Another crucial factor in creating realistic vocal fold models is layering, as vocal folds consist of multiple layers of varying softness, which determine the pattern of flow-induced vibration and the frequency at which vibration is possible.
In this study, we created a typical vocal fold model. We used the common geometry provided by Scherer5, which represents typical dimensions for male vocal folds with 17 mm length according to Zhang6 and consists of three layers: one layer for the vocalis muscle (body layer), one for the entire mucosal layer (cover layer), and one for the epithelium. This structure can be seen in the coronal cross-section view in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66222/66222fig01v2.jpg" /> 
Figure 1: Coronal cross-section of the larynx modules. Coronal cross-section of the larynx modules illustrating the widest width of the vocal folds (8.5 mm). Each vocal fold comprises a body layer, a cover layer, and an epithelium layer. This figure has been modified from13. Reproduced from H&#228;sner, P., Prescher, A., Birkholz, P. Effect of wavy trachea walls on the oscillation onset pressure of silicone vocal folds. J Acoust Soc Am.149 (1), 466-475 (2021) with the permission of the Acoustical Society of America. <a href="https://www.jove.com/files/ftp_upload/66222/66222fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Other publications use partially only one layer7, two layers without epithelium layer2 or model the mucosa with multiple layers3. Usually, the layers are cast from the inside out, i.e., starting with the deepest layer. The epithelium, which is very thin with 30 &#181;m thickness, is cast at the end over the entire body to envelop it with a sturdy skin8.
The cover layer in the model is the softest part, with Young&#39;s modulus of about 1.1 kPa9. For the body layer, the approximate Young&#39;s modulus in the transverse direction using in vitro measurements10 is 2 kPa. In vivo, the Young&#39;s modulus of the thyroarytenoid muscle may be higher due to the presence of fibers in the longitudinal direction as well as the possible tensing of the muscle. To achieve this extremely low Young&#39;s modulus, it is necessary to add a high amount of silicone oil to the silicone mixture (approximately 72%). However, the manufacturer strongly advises against using an oil proportion greater than 5%. In general, the addition of silicone oil to the elastomer is intended to increase flow and drip time, as well as reduce the shrinkage of the cured silicone polymer. It helps the silicone to cure more uniformly, thereby reducing stress in the material. Its purpose is to optimize the moldability and properties of the cured material, rather than to increase its softness, although this is also a consequence. This is because silicone oil is chemically inert, meaning that it cannot polymerize itself and is not integrated into the network of the silicone polymer11. Instead, it remains as a liquid phase in the polymer matrix, weakening the polymer structure at higher levels and potentially causing it to dissolve out of the cured material and adhere to the surface. As a result, other negative properties such as curing disorders, uneven vulcanization, chemical shrinkage, and brittleness are possible. Vocal fold models with high silicone oil contents were investigated with regard to aging and reproducibility, and it was found that there is a high variability in the properties of different models and a change in their properties over time11.
When producing vocal fold models in the conventional way7,12, the stickiness of the epithelial layer can be a problem as it can affect the homogeneity of vibration and lead to rupture of the epithelium. Although the silicone used to make the epithelium is undiluted, it can be assumed that the oil that leaks from the neighboring mucosa layer has similar effects on the silicone as if it had been diluted. The problem of stickiness was addressed by adding various powders such as talcum or carbon powder as an intermediate layer between the mucosa and the epithelial layer12. This approach may have been successful because the oil was partially absorbed by the powder and, as a result, the stickiness of the epithelial surface could be reduced.
In this publication, we show that the problem of stickiness can be circumvented by a slight modification of the process of vocal fold manufacturing. By changing the order of layering and starting with the undiluted epithelial silicone (so-called closed silicone), non-sticky super-soft vocal fold models can be produced. This change involves unusual types of molds and methods that are best presented and explained in the form of a video. In this paper, we describe our manufacturing process in detail and demonstrate how the properties of the vocal fold models can be characterized in an application.

<table><tbody><tr><td>3D Printer</td><td>ULTIMAKER</td><td></td><td>Type S5</td></tr><tr><td>3D Printing software</td><td>ULTIMAKER CURA</td><td></td><td>Version 5.2.2</td></tr><tr><td>CAD Software</td><td>Autodesk Inventor&amp;nbsp;</td><td></td><td>Version 2023</td></tr><tr><td>High Speed Camera</td><td>XIMEA GmbH</td><td></td><td>MQ013CG-ON</td></tr><tr><td>PLA+ 3D Printer Material&amp;nbsp;</td><td>eSun</td><td>none</td><td>white</td></tr><tr><td>Primary silicone</td><td>KauPo Plankenhorn</td><td>09301-005-000041</td><td>EcoFlex 00-30</td></tr><tr><td>Release Agent</td><td>KauPo Plankenhorn</td><td>09291-006-000001</td><td>UTS Universal</td></tr><tr><td>Secondary silicone</td><td>KauPo Plankenhorn</td><td>09301-005-000181</td><td>DragonSkin NV10</td></tr><tr><td>Silicone Thinner</td><td>KauPo Plankenhorn</td><td>09301-010-000002</td><td></td></tr><tr><td>Tougth PLA 3D Printer Material&amp;nbsp;</td><td>BASF</td><td></td><td>black</td></tr></tbody></table>

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.

The fabricated vocal fold model was integrated into the measurement setup depicted in Supplementary Figure 3 at the vocal folds position. The setup, extensively detailed in a previous publication13, comprises a multi-stage controllable airflow source that stimulates the vocal fold models into oscillation, along with an array of measuring instruments that record data such as sound pressure, static pressure at specific positions, and volume velocity. For the measurements, the airflow gradually increased until the vocal fold model began to oscillate. Subsequently, the air pressure was elevated by 200 Pa above the onset pressure to achieve a stable and robust oscillation. An additional high-speed camera was added and placed above the vocal tract model, capturing the vocal fold oscillation movements at a maximum frame rate of 2304 frames per second.
A lamp integrated within the lung emits light through the subglottal tract, causing the glottis to appear white. Figure 2 depicts two series of oscillation images, each consisting of six frames, illustrating a typical close-open-close cycle. The upper row (Figure 2A) displays the oscillation of vocal folds manufactured using the presented method, while the lower row (Figure 2B) demonstrates an extreme example of a conventional vocal fold model, created during the preliminary work13, incapable of generating stable oscillation due to its sticky surface. For the latter, the surface stickiness causes the glottis to open at the anterior and posterior ends first, and the central part opens later. The model&#39;s surface is already slightly damaged at a specific point due to adhesion.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66222/66222fig02.jpg" /> 
Figure 2: Sequence of individual frames captured by the high-speed camera. Sequence of individual frames captured by the high-speed camera, showcasing a close-open-close cycle of vocal fold vibration. (A) Vocal folds fabricated using the presented method. (B) Vibration of a conventional vocal fold model with sticky surface. <a href="https://www.jove.com/files/ftp_upload/66222/66222fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 and Figure 4 show the time functions of the glottal area of the proposed model and the conventional (sticky) model, respectively. The area waveform (left part in each of the figures) was computed using the GlottalImageExplorer software14 from the available image sequences. The right parts of the figures show the magnitude spectra of the time functions to indicate their degree of periodicity. The fundamental frequency was extracted from the time functions using the Praat software15. It is evident from Figure 3 that the proposed vocal fold model shows a stable oscillation over the selected duration, enabling the accurate calculation of the fundamental frequency. In contrast, Figure 4 displays an atypical and chaotic glottal area function with inconsistent minima and maxima, along with various artifacts. The extraction of the fundamental frequency becomes challenging or even unfeasible in this scenario.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66222/66222fig03.jpg" /> 
Figure 3: Area waveform for a vocal fold model fabricated using the presented method. Representation of the area waveform obtained from the high-speed camera image data using (A) the GlottalImageExplorer, as well as (B) the derived magnitude spectrum for a vocal fold model fabricated using the presented method. <a href="https://www.jove.com/files/ftp_upload/66222/66222fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66222/66222fig04.jpg" /> 
Figure 4: Area waveform for a vocal fold model with sticky surface. Representation of the area waveform obtained from the high-speed camera image data using (A) GlottalImageExplorer, as well as (B) the magnitude spectrum using a conventional vocal fold model with sticky surface. <a href="https://www.jove.com/files/ftp_upload/66222/66222fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: List of essential components for manufacturing a vocal fold half. List of essential components for manufacturing a vocal fold half. 1 - Support structures for one vocal fold half, 2a-c - Mold components for crafting the body layer, 3a-c - Mold components for crafting the cover layer, 4 - Support structures for attachment. <a href="https://www.jove.com/files/ftp_upload/66222/fig-sm-requiered-parts.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Schematic depiction of mold assembly. Schematic depiction of mold assembly. Left - Mold for creating the body layer, Right - Mold for creating the cover layer. Labels correspond to the parts list in Supplementary Figure 1. <a href="https://www.jove.com/files/ftp_upload/66222/fig-sm-construction-scheme.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Complete setup of the measurement system. Complete setup of the measurement system. This figure has been modified from13. Reproduced from H&#228;sner, P., Prescher, A., Birkholz, P. Effect of wavy trachea walls on the oscillation onset pressure of silicone vocal folds. J Acoust Soc Am.149 (1), 466-475 (2021) with the permission of the Acoustical Society of America. <a href="https://www.jove.com/files/ftp_upload/66222/fig-sm-measurement-setup.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 1: Support structures for one vocal fold half. This is the file to produce vocal-fold-positiv. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-vocal-fold-positiv.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 2: Mold component 1 for crafting the body layer. This is the file to produce vocalis_mold-main-part. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-1_vocalis_mold-main-part.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 3: Mold component 2 for crafting the body layer. This is the file to produce vocalis_mold-cap. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-1_vocalis_mold-cap.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 4: Mold hull for crafting the body layer to avoid leakage of silicone. This is the file to produce vocalis_mold-hull. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-1_vocalis_mold-hull.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 5: Mold component 1 for crafting the cover layer. This is the file to produce mucosa_mold-main-part. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-2_mucosa_mold-main-part.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 6: Mold component 2 for crafting the cover layer. This is the file to produce mucosa_mold-back. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-2_mucosa_mold-back.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 7: Mold hull for crafting the cover layer to avoid leakage of silicone.&#160;This is the file to produce mucosa_mold-hull. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-2_mucosa_mold-hull.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 8: Support structures for attaching the vocal fold halves. This is the file to produce measurement-pressure-tap-adapter.&#160;Support structures for attaching the vocal fold halves including pressure measuring tap. <a href="https://www.jove.com/files/ftp_upload/66222/sec2-4_measurement-pressure-tap-adapter.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models at JoVE.com

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.

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 ...

manufacturing-process-for-non-adhesive-super-soft-vocal-fold-models

Nanyang Technological University

Research

JoVE Journal

Engineering

731 Views.  Technische Universität Dresden. 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.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Bioengineering

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 ...

Fabrication Process of Silicone-based Dielectric Elastomer Actuators

This contribution demonstrates the fabrication process of dielectric elastomer transducers (DETs). DETs are stretchable capacitors consisting of an elastomeric dielectric membrane sandwiched between two compliant electrodes. The large actuation strains of these transducers when used as actuators (over 300% area strain) and their soft and compliant nature has been exploited for a wide range of applications, including electrically tunable optics, haptic feedback devices, wave-energy harvesting, deformable cell-culture devices, compliant grippers, and propulsion of a bio-inspired fish-like airship. In most cases, DETs are made with a commercial proprietary acrylic elastomer and with hand-applied electrodes of carbon powder or carbon grease. This combination leads to non-reproducible and slow actuators exhibiting viscoelastic creep and a short lifetime. We present here a complete process flow for the reproducible fabrication of DETs based on thin elastomeric silicone films, including casting of thin silicone membranes, membrane release and prestretching, patterning of robust compliant electrodes, assembly and testing. The membranes are cast on flexible polyethylene terephthalate (PET) substrates coated with a water-soluble sacrificial layer for ease of release. The electrodes consist of carbon black particles dispersed into a silicone matrix and patterned using a stamping technique, which leads to precisely-defined compliant electrodes that present a high adhesion to the dielectric membrane on which they are applied.

This manuscript shows the fabrication process for the manufacture of dielectric elastomer soft actuators based on silicone membranes. The three key stages of production are presented in detail: blade casting of thin silicone membranes; pad printing of compliant electrodes; and the assembly of all the components.

This contribution demonstrates the fabrication process of dielectric elastomer transducers (DETs). DETs are stretchable capacitors consisting of an ...

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 ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Manufacture of a Multi-Purpose Low-Cost Animal Bench-Model for Teaching Tracheostomy

Tracheostomy is one of the most frequent procedures, performed through various techniques in the intensive care unit and emergency situations. Despite this, there is a lack of training on this procedure that affects its outcome, which is also dependent on operator's dexterity. Here, we take the specific training and simulation into consideration. This article aims to describe every step of the manufacture of a new multi-purpose low-cost animal bench-model, with the support of video and images, and to obtain an opinion about the quality of this model by administering a questionnaire to professionals with experience in the procedures. 
Ten experts in the technique were enrolled. The model scored an average of 3.45/5 for its anatomical realism; 4.75/5 for its usefulness as a training tool for simulation courses and assessments. The time necessary to build the model was 15 minutes, and the cost amounted to 10&#8364;. The animal bench-model was considered a very useful simulator for tracheostomy training and assessments. Therefore, it could be used as a tool for medical courses and residencies.

This article illustrates every step of the manufacture of a new multi-purpose low-cost animal bench-model for subglottic airway access management. All the procedures are shown in the video. The model's realism and its suitability for training the given clinical maneuvers were assessed by independent senior otolaryngologists and anesthesiologists.

Tracheostomy is one of the most frequent procedures, performed through various techniques in the intensive care unit and emergency situations. Despite ...

Medicine

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

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.

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.