The authors gratefully acknowledge Grants R03DC8200, R01DC9616, and R01DC5788 from the National Institute on Deafness and Other Communication Disorders for support of synthetic model development.

The fabrication sequence (see Fig. 1) consists of making molds for vocal fold model layers, sequentially casting silicone layers, and mounting the models for testing. The model has four distinct layers: body, ligament, superficial lamina propria, and epithelium, in addition to a single fiber. A backing layer is added to facilitate the accurate placement of individual layers to the vocal fold model. The model geometric parameter definitions are shown in Fig. 2, with parameter values for the current model given in Table 1. In the following sections, different silicone mixing ratios are specified for the different layers; these produce material properties that are similar to those reported for human vocal fold tissue in the small strain regime 13 (see Table 2).
1. Mold fabrication and preparation
<ol>
	<li>Create solid models of three vocal fold layers: superficial lamina propria, ligament, and body layers. This is typically done by creating 3D computer-aided design (CAD) models with the desired geometries, exporting the CAD models as stereolithography (STL) files, and sending the STL files to a custom machine shop for rapid prototyping.</li>
	<li>Create a box-shaped mold form using thin pieces of acrylic material. Approximate dimensions (not critical) are 2.54 cm high &times; 5.72 cm wide &times; 6.35 cm deep. Make the bottom of the form by adhering it to a flat acrylic plate. Seal all interior edges with vacuum grease.</li>
	<li>Place a small amount of vacuum grease on the lateral side of the solid model of desired geometry (i.e., the body, the ligament, or the superficial lamina propria). Press model into bottom of mold form cavity, vacuum grease side down, so that the vacuum grease holds the part in place. Liberally coat mold form and solid model with release agent. Using a paint brush, ensure release agent reaches into all corners of the mold form cavity.</li>
	<li>Mix 10 parts A and one part B of Smooth-Sil 950 platinum silicone rubber (parts measured by weight) in a container that has sufficient room for expansion. To remove air bubbles place mold form with uncured silicone rubber in a vacuum chamber and reduce pressure (e.g., to around 26 inches Hg below atmospheric pressure) for approximately three minutes (or more or less as necessary). Remove degassed silicone from chamber and pour into mold form cavity. Place mold with uncured silicone into vacuum chamber and degas again. Remove from vacuum chamber and place on level surface. Allow to cure for 24 hours and remove mold from mold form.</li>
	<li>Repeat steps 1.1 through 1.4 to create molds for each of the superficial lamina propria, ligament, and body layers.</li>
	<li>Cut ligament layer mold at the center of the medial surface in the anterior-posterior direction with a straight razor to allow for fiber insertion.</li>
</ol>
2. Casting of each layer
<ol>
	<li>Body Layer: Apply thin layer of release agent to body mold cavity with paint brush. Mix one part B and one part A of Ecoflex 00-30 Supersoft Platinum Silicone (by weight). Add one part Silicone Thinner (by weight) to reduce the eventual cured stiffness of the material. Mix together for 30 seconds and place in vacuum chamber for one minute to remove entrapped air. Remove mixture from vacuum and pour into the body mold cavity, but do not fill to the top of the entire mold cavity. Place in oven at 250 &deg;F for 30 minutes. Remove from oven and cool.</li>
	<li>Backing: Mix one part B and one part A of Dragon Skin and add one part Silicone Thinner (by weight). Mix vigorously for 30 seconds, place in vacuum for 1 minute, and pour into the body mold cavity until full. Place in oven at 250 &deg;F for 30 minutes. Remove mold from oven and cool. Remove model from mold, allow to cool to room temperature, and remove any release agent on the surface of the body layer with paper towel.</li>
	<li>Ligament Layer: Apply thin layer of release agent on ligament mold cavity surface with paint brush. Place a 30 cm thread in the mold by pushing it into the cut from the straight razor. Thoroughly mix one part B and one part A of Ecoflex 00-30 and four parts of Silicone Thinner (by weight). Place in vacuum chamber to remove air bubbles and pour mixture into ligament mold cavity.</li>
	<li>Ligament Layer (continued): Press body-backing model (from Steps 2.1.1 and 2.1.2) into the ligament mold cavity. Begin insertion at one side and gently move to the other so that the model pushes the excess uncured silicone and air bubbles out of the mold cavity. If air bubbles are present, remove the model from the mold cavity, refill with uncured silicone, and repeat pressing model into the mold. Place mold in oven for 30 minutes, remove, and cool to room temperature. Remove model from mold. Remove excess release agent with paper towel.</li>
	<li>Superficial Lamina Propria Layer: Apply thin layer of release agent on superifical lamina propria (SLP) mold cavity surface with paint brush. Mix one part B, one part A of Ecoflex 00-30, and 8 parts Silicone Thinner by weight. Vacuum as done previously and pour into SLP mold cavity. Use the same process described in Step 2.1.4 to insert the ligament-body-backing model into the superficial lamina propria mold cavity. Place in an oven at 250 &deg;F and cure for one hour. Remove from oven and allow to cool. Remove model slowly and with extreme care so that the superficial lamina propria remains intact.</li>
	<li>Epithelium Layer: Place vocal fold model on a flat surface with the backing down. Remove support material with a straight razor. Suspend threads in air by attaching them to an object of greater height than the model. Mix one part B and one part A of Dragon Skin with one part of Silicone Thinner, mix, vacuum, then pour over the model and allow to cure for one hour. Repeat the process to create a thicker layer. Remove excess material with a straight razor.</li>
	<li>Optional: If each layer is desired to be a different color (for visible inspection of different layers), add dye to part B of either the Ecoflex or Dragon Skin during the mixing process.</li>
	<li>Optional: If material property data will be collected, create tensile and rheological specimens simultaneously with fabrication of each model layer. Do this by pouring extra uncured material into release agent-treated molds of desired material property specimen shape and size.</li>
	<li>Optional: If measurements of layer thickness are desired, cut a cross section of the model with a straight razor and inspect with microscope.</li>
</ol>
3. Final model preparation for testing
<ol>
	<li>Mount each completed vocal fold model into an acrylic mounting plate by first applying a thin layer of silicone glue on the back (lateral) and side (anterior-posterior) model surfaces. Insert model into recessed cut of mounting plate. Align the model medial surface with the top of the acrylic plate. Wipe away excess glue. Allow glue to cure for one hour.</li>
	<li>Apply talc powder to the model surface to reduce surface tackiness.</li>
	<li>For medial surface tracking use a fine-point Sharpie pen to mark dots on the model. Best results occur if marking is done after application of talc powder.</li>
	<li>Place long bolts through holes of the mounting plate with the threaded ends pointing toward the model to which the existing model will be paired. Lay threads over the bolts. Put closed cell foam over the bolts to close any air gaps.</li>
	<li>Pair this prepared model with another vocal fold model that has been similarly mounted to an acrylic holder using Steps 3.1 and 3.2. Tighten screws to compress the foam and bring the medial surfaces together until the desire pre-vibratory gap is reached. Ensure both sets of threads are placed over the bolts and extend outward from the acrylic plates in the anterior-posterior direction.</li>
	<li>Mount vocal fold pair on air supply tube.</li>
	<li>Tie the anterior threads together to form a loop. Repeat for the posterior threads. Hang desired weight on the loops simultaneously.</li>
	<li>Models are now ready for testing and data collection.</li>
</ol>
4. Representative Results
Vibratory response data from one model created using this fabrication process are as follows; these results are typical. With tension of approximately 31 g applied to the fibers, the onset pressure was 400 Pa. At a subglottal pressure of 10% above onset pressure (440 Pa), the model vibrated at 115 Hz with a glottal flow rate of 210 ml/s. These values are in good agreement with values reported for those of humans (Table 3). Using high-speed videokymography to analyze model motion showed evidence of a phase difference between the superior and inferior margins, i.e., the superior margin concealed the inferior margin during the open phase of the vibration period (Fig. 3). Trajectories extracted from stereo images of the dots applied to the medial and inferior surfaces of the vocal fold model showed that the model exhibited an alternating convergent-divergent profile that is typical of human phonation, a mucosal wave-like motion, and a lower inferior-superior motion than in previous models (Fig. 4).
<img alt="Table 1." src="/files/ftp_upload/3498/3498table1.jpg" />
Table 1. Model geometric parameter values.
<img alt="Table2." src="/files/ftp_upload/3498/3498table2.jpg" />
Table 2. Mixture ratios by weight and resulting Young's modulus of the individual sections of the vocal fold model. EF and DS designate silicone made from Ecoflex and Dragon Skin, respectively 14.
<img alt="Table 3." src="/files/ftp_upload/3498/3498table3.jpg" />
Table 3. Comparison between human and synthetic vocal fold vibratory responses.
<img alt="Figure 1." src="/files/ftp_upload/3498/3498fig1.jpg" />
Figure 1. Synthetic vocal fold model fabrication process. CAD-derived solid models (left panel) are used to create molds (center panel) for each layer. Each layer is then cast, beginning with the body layer and ending with the epithelium layer (right panel, with each layer "peeled back" for visibility). After fabrication, models are mounted to acrylic plates for testing.
<img alt="Figure 2." src="/files/ftp_upload/3498/3498fig2.jpg" />
Figure 2. Synthetic vocal fold model cross section. Distinct body, superficial lamina propria, ligament, and epithelium layers are shown. Parameters define vocal fold model geometry. This figure is scaled for clear representation of geometric definitions. Application of the parameter values given in Table 1 will result in a slightly different shape than what is shown here.
<img alt="Figure 3." src="/files/ftp_upload/3498/3498fig3.jpg" />
Figure 3. High-speed kymogram of model vibration. Estimates for the location of the superior and inferior margins are shown in colored dotted lines. Phase differences between the inferior and superior margins are evident.
<img alt="Figure 4." src="/files/ftp_upload/3498/3498fig4.jpg" />
Figure 4. Medial surface profile of synthetic vocal fold model in a hemilarynx arrangement, captured at two different instances of time while vibrating. Ink markers were placed on the medial surface (as shown in the right image), imaged using two synchronized high-speed cameras, and tracked over the vibratory cycle. The left plot shows a convergent glottis during the opening phase and the right plot shows a divergent glottis during the closing phase.

<ol>
	<li>Hirano, M., Kakita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cover-body+theory+of+vocal+fold+vibration.">Cover-body theory of vocal fold vibration.</a> Speech Science: Recent Advances. , 1-46 (1985).</li><li>Pickup, B. A., 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+asymmetric+stiffness+on+the+structural+and+aerodynamic+response+of+synthetic+vocal+fold+models.">Influence of asymmetric stiffness on the structural and aerodynamic response of synthetic vocal fold models.</a> Journal of Biomechanics. 42 (14), 2219-2225 (2009).</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=Vibration+in+a+self-oscillating+vocal+fold+model+with+left-right+asymmetry+in+body-layer+stiffness.">Vibration in a self-oscillating vocal fold model with left-right asymmetry in body-layer stiffness.</a> Journal of the Acoustical Society of America. 128 (5), EL279-EL285 (2010).</li><li>Popolo, P. S., Titze, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualification+of+a+Quantitative+Laryngeal+Imaging+System+Using+Videostroboscopy+and+Videokymography.">Qualification of a Quantitative Laryngeal Imaging System Using Videostroboscopy and Videokymography.</a> Annals of Otology, Rhinology &amp; Laryngology. 117 (6), 4014-4412 (2008).</li><li>Thomson, S. L., Mongeau, L., Frankel, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerodynamic+transfer+of+energy+to+the+vocal+folds.">Aerodynamic transfer of energy to the vocal folds.</a> Journal of the Acoustical Society of America. 118 (3), 1689-1700 (2005).</li><li>Neubauer, J., Zhang, Z., Miraghaio, R., Berry, D. 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The authors have nothing to disclose.

This method of fabricating synthetic vocal fold models yields models that exhibit vibratory behavior similar to that of human vocal folds. The multi-layer concept results in significant advantages over previous one- and two-layer model designs 3,6,8,15, in terms of reduced onset pressure and improved model motion (convergent-divergent profile during oscillation, mucosal wave-like motion, and reduced inferior superior displacement). The method presented here is demonstrated on a somewhat idealized model in term...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>High Vacuum Grease</td>
 <td>Dow Corning</td>
 <td>01018817</td>
 <td></td>
 </tr>
 <tr>
 <td>Pol-Ease 2300</td>
 <td>Polytek</td>
 <td>Pol-Ease2300-1</td>
 <td>Release agent</td>
 </tr>
 <tr>
 <td>Smooth-Sil 950</td>
 <td>Smooth-On</td>
 <td>Smooth-Sil 950</td>
 <td>Mold making material</td>
 </tr>
 <tr>
 <td>Vacuum Pump</td>
 <td>Edwards</td>
 <td>E2M2</td>
 <td></td>
 </tr>
 <tr>
 <td>Vacuum Chamber</td>
 <td>Kartell</td>
 <td>230</td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure Gage</td>
 <td>Marsh Bellofram</td>
 <td>11308252A</td>
 <td></td>
 </tr>
 <tr>
 <td>Straight Razor</td>
 <td>Husky</td>
 <td>008-045-HKY</td>
 <td></td>
 </tr>
 <tr>
 <td>Ecoflex 00-30</td>
 <td>Smooth-On</td>
 <td>Ecoflex 00-30</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone Thinner</td>
 <td>Smooth-On</td>
 <td>Silicone Thinner</td>
 <td></td>
 </tr>
 <tr>
 <td>Dragon Skin</td>
 <td>Smooth-On</td>
 <td>Dragon Skin 10 FAST</td>
 <td></td>
 </tr>
 <tr>
 <td>Thread</td>
 <td>Omega</td>
 <td>OmegaCrys</td>
 <td>Use only clear fibers</td>
 </tr>
 <tr>
 <td>Silicone Dye</td>
 <td>Smooth-On</td>
 <td>Silc Pig Black</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone Glue</td>
 <td>Smooth-On</td>
 <td>Sil-Poxy</td>
 <td></td>
 </tr>
 <tr>
 <td>Talc Powder</td>
 <td>Western Family</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>

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.

Watch this Scientific Journal Video about Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication at JoVE.com

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

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 ...

synthetic-multi-layer-self-oscillating-vocal-fold-model-fabrication

Research

JoVE Journal

Bioengineering

13.9K Views.  Brigham Young University. 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.

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

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

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 ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

Versatile Biomolecular Condensates from Amphiphilic DNA Nanostars or C-Stars

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Author Spotlight: Developing Synthetic Cells from Programmable Amphiphilic DNA Nanostructures

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be ...

Methods Article

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