This work is supported by the National Science Foundation, Grant No. CBET-1236351 and GW Center for Biomimetics and Bioinspired Engineering (COBRE).

In this work, a wall-mounted prolate hemispheroid, i.e. a model vocal fold polyp, is positioned on the test section floor of a suction type wind tunnel with a 5:1 contraction ratio. Unsteady, three-dimensional flow separation and its effect on the wall pressure loading are investigated using oil-flow visualization, wall pressure measurements, and particle image velocimetry. The unsteady pressure measurements are acquired using a sixteen channel scanning pressure transducer with piezoresistive pressure sensors. The pressure sensors have a frequency response of 670 Hz. Static pressure taps formed from stainless steel tubulations are flush-mounted upstream and downstream of the model vocal fold polyp to facilitate the surface pressure measurements and short-plumbed to the scanning pressure device. Oil-flow visualization and surface pressure measurements cannot be acquired simultaneously because oil would flow into the pressure taps causing fouling.
The following section provides the protocol for setting up and acquiring oil-film visualization and surface pressure measurements around a wall mounted prolate hemispheroid. Although phase-averaged and time resolved particle image velocimetry measurements are being acquired, the PIV acquisition is not included in this protocol. The authors suggest the references by Raffel et al.36 and Adrian and Westerweel39 for an in depth understanding of PIV experimental setup, data acquisition, and data processing.
1. Generate Protuberance (i.e.&#160;Model Polyp)
<ol>
	<li>Build a three-dimensional computer-aided design (CAD) model with the desired geometry. Generate the model vocal fold polyp as a prolate hemispheroid measuring 5.08 cm long, 2.54 cm wide, and 1.27 cm tall. Mount a 2.54 cm square base that is 0.64 cm thick to the bottom of the model vocal fold polyp. This base will be used to anchor the model to the test section floor.</li>
	<li>Export the 3D CAD model as a stereolithography (STL) file. The STL file format generates the model surface as a series of triangles. Choose an adequate resolution to ensure a smooth surface on the model polyp. A resolution of at least 600 dots/in is recommended.</li>
	<li>Upload the STL file into the appropriate software and print the STL file using a high resolution three-dimensional printer or rapid prototyper with a build layer resolution of at least 20 &#956;m.</li>
	<li>The wind tunnel test section is approximately 30.48 cm x 30.48 cm x 121.92 cm with a removable bottom plate as shown in Figure 1. Mill a 2.54 cm square hole approximately 0.85 cm deep into the wind tunnel test section floor removable plate to mount the model vocal fold polyp for testing. The hole should be located in the center of the test section width and be located at the desired downstream location for testing.</li>
</ol>
2. Oil-flow Visualization Preparation
<ol>
	<li>In order to prepare the test section, cover the test section surface inside of the wind tunnel with white adhesive paper. Carefully place and smooth the adhesive paper to ensure that the test section floor has no bumps due to air bubbles or creases in the adhesive paper. Cut a hole in the adhesive paper above the square hole in the test section floor for the model polyp anchor to attach to the test section wall.</li>
	<li>Insert the protuberance (model vocal fold polyp) into the anchor position to prepare for testing. See Figure 1.</li>
	<li>Mount a high resolution camera above the wind tunnel test section. Focus the camera for the chosen field of view including the model polyp and surrounding test section area. Set the camera acquisition parameters for testing. A video setting should be used to capture the transient portion of the oil-flow visualization or if unsteady or pulsatile flows are of interest.</li>
	<li>Prepare the flow-visualization oil mixture by combining baby oil, copy toner powder, and kerosene in a 7:1:2 ratio by volume. For example: combine 35 ml&#160;baby oil, 5 ml&#160;copy toner powder, and 10 ml&#160;kerosene. Mix the baby oil and toner powder together in a container and stir until the toner is completely dissolved. Then add the kerosene and mix well.</li>
	<li>Transfer the mixture to a spray bottle for easy application to the test section surface.</li>
</ol>
3. Oil-flow Visualization Measurements
<ol>
	<li>Clean and dry the test section surface before each application of the oil mixture.</li>
	<li>Use the spray bottle filled with the oil mixture to spray a thin, even layer of fluid over the section of interest. A thin, even oil mixture layer is important for producing proper oil-film visualization images.</li>
	<li>Initiate the image or video acquisition on the camera. Begin the camera acquisition before the wind tunnel is powered on in order to capture the initial transient oil mixture motion.</li>
	<li>Set the suction wind tunnel to the desired velocity. The oil mixture will begin to flow along the test section surface.</li>
	<li>Once the oil mixture stops flowing and has reached a steady state (i.e. the patterns are stationary), or when the desired time has elapsed, stop the camera recording and power down the wind tunnel. 
	Note: Video 1 displays the oil-mixture flowing until a steady state is reached and the skin friction pattern becomes stationary. In the video the flow is moving from left to right.</li>
</ol>
4. Surface Pressure Measurement Preparation
<ol>
	<li>Prepare the test section floor surface (removable plate) by drilling holes for mounting stainless steel tubulations (0.16 cm outer diameter and 2.54 cm long) into the test section floor to build static pressure taps. Starting at the midline of the anchor position of the prolate hemispheroid, drill the holes on a grid that spans 8.89 cm in the spanwise direction and 22.86 cm downstream with 1.27 cm spanwise grid spacing and 2.54 cm downstream grid spacing (see Figure 1). The stainless steel tubulations have a bulge on one end for attaching flexible tubing and are straight on the other end for mounting. 
	Note: The static pressure taps can be positioned at closer intervals for a finer grid of pressure acquisition locations.</li>
	<li>Mount the tubulations surrounding the anchor position of the wall mounted prolate hemispheroid (i.e. model vocal fold polyp) in the desired configuration on the test section floor to prepare for testing. The tubulations should be mounted flush with the test section floor.</li>
	<li>Attach pieces of short flexible tubing (6.35 cm length, 0.159 cm inner diameter, 0.475 cm outer diameter clear polyvinyl chloride tubing) from the mounted stainless steel tubulations to the scanning pressure transducer measurement ports. The scanning pressure transducer has sixteen pressure ports.</li>
</ol>
5. Surface Pressure Measurement Acquisition
<ol>
	<li>Connect the scanning pressure transducer to a computer and configure the acquisition parameters using the scanning pressure transducer software. Set the acquisition software to acquire data at 500 Hz for the desired duration of data acquisition. 
	Note: Data was acquired at the maximum sampling rate of the scanning pressure transducer, 500 Hz, due to the small pressure variations at low oscillation frequencies.</li>
	<li>Set the suction wind tunnel to the desired velocity.</li>
	<li>Begin pressure measurement acquisition. The pressure measurements may be acquired simultaneously with any desired flow diagnostic technique (e.g. PIV, laser Doppler anemometry, hot wire anamometry, etc.)</li>
</ol>

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The authors have nothing to disclose.

Understanding the formation and propagation of vortical structures from a geometric protuberance&#160;and their subsequent effect on the aerodynamic loadings that drive vocal fold dynamics, is necessary to provide insight and models in order to advance the treatment of vocal fold polyps and nodules. The variations in aerodynamic loadings caused by the model polyp in this experiment are expected to contribute to irregular vocal fold dynamics observed in patients with polyps13,41. Future work includes investigat...

The vocal folds are two bands of tissue that stretch across the vocal airway. Voiced speech is produced when a critical lung pressure is achieved, forcing air through adducted vocal folds. The vocal folds are composed of many layers of tissue and are often represented by a simplified two-layer body-cover system1. The extracellular matrix, which makes up the majority of the cover layer, is composed of collagen and elastin fibers, providing nonlinear stress-strain characteristics, which are important to the proper motion of the vocal folds1,2. Aerodynamic forces impart energy to the tissue of the vocal folds and excite self-sustained oscillations3. As the vocal folds oscillate, the opening between them, referred to as the glottis, forms a temporally-varying orifice that transitions from a convergent to a uniform and then to a divergent passage before closing and repeating the cycle4,6. Frequencies of vibration for normal speech typically span 100-220 Hz in males and females respectively, creating a pulsatile flow field that passes through the glottis7. The fluid-structure energy exchange process for normal speech has been studied extensively8-12; however, the disruption of this process for some pathologies is not well understood. Pathological conditions of the vocal folds can result in dramatic changes in their dynamics and affect the ability to generate voiced speech.
Polyps and nodules are geometric abnormalities that form on the medial surface of the vocal folds. These abnormalities can affect a patient&#39;s ability to communicate13. Nevertheless, only recently has the disruption of the flow field due to a geometric protuberance such as a polyp been considered14. That study showed that the &#34;normal&#34; fluid-structure energy-exchange process of speech was drastically altered, and that the modification of the flow field was the most likely reason for the severe degradation of vocal quality in patients with polyps and nodules. No comprehensive understanding of the flow structures produced by three-dimensional flow separation from a polyp in pulsatile flow has been established. The generation and propagation of vortical structures from a polyp, and their subsequent impact on the aerodynamic loadings that drive vocal fold dynamics is a necessary critical component to advance surgical remediation of polyps in patients.
While flow separation from a wall mounted hemispheroid in steady flow has been investigated15-23, surprisingly, there is little information regarding unsteady three-dimensional flow separation from a hemispheroid on a wall subject to pulsatile or unsteady flow conditions as are found in speech. The seminal work of Acarlar and Smith15 provided an analysis of the three-dimensional coherent structures generated by steady flow over a wall mounted hemispheroid within a laminar boundary layer. Acarlar and Smith identified two types of vortical structures. A standing horseshoe vortex was formed upstream of the hemispheroid protuberance and extended downstream of the protuberance on either side. Additionally, hairpin vortices were shed periodically from the wall mounted hemispheroid into the wake. The complex motion and progression of the hairpin vortices was investigated and described in detail.
Flow over a smoothly contoured axisymmetric hill has been previously studied in which both surface static pressure measurements and surface oil visualization were acquired on and downstream of the bump within a turbulent shear flow. Oil-film techniques enable the visualization of skin friction lines, high and low velocity regions, and separation and attachment points within a surface flow, and are useful to investigate the wake of a wall-mounted object. For this technique, the surface of interest is coated with a thin film of an oil-base and fine powder pigment (i.e. lampblack, graphite powder, or titanium dioxide) mixture. At the desired flow conditions, frictional forces cause the oil to move along the surface causing the pigment powder to be deposited in streaks. Critical or singularity points, locations where the shear stress is zero or two or more components of the mean velocity are zero, can be classified from the resulting skin friction line pattern as saddle points or nodal points24-26.
For the hill geometry, no singularity caused by separation was found upstream; this was attributed to the smoothly ascending contour of the bump, which did not generate the adverse pressure gradient that occurs with a hemispheroid protuberance. Consequently, the flow was found to accelerate until the pinnacle of the bump after which, unsteady saddle-focus separation points developed shortly past the bump centerline, as would be expected from the formation of a hairpin vortex27,28. In a study using similar experimental techniques with a different wall-mounted geometry, oil-film visualization around a surface-mounted cube in steady flow performed by Martinuzzi and Tropea29 displayed two clear skin friction lines upstream of the object. The first skin friction line corresponded to the primary separation line caused by the adverse pressure gradient and the second skin friction line marked the time-averaged location of the horseshoe vortex. Surface pressure measurements performed upstream of the object showed a local minimum along the horseshoe vortex line and a local pressure maximum between the primary separation and horseshoe vortex lines. Similar upstream separation lines are formed with other surface-mounted geometries including a circular cylinder, pyramid, and cone29-31. Surface visualization downstream of wall-mounted objects typically displays two foci caused by the recirculation region behind the object30. Two vortices are generated at the foci positions and correspond to the &#34;arch-type&#34; or hairpin vortex seen in the wake of a wall-mounted hemispheroid32.
Particle image velocimetry (PIV) has been previously used to study the flow downstream of synthetic vocal fold models33-35. PIV is a noninvasive visualization technique which images flow tracer particle movement within a plane at to capture spatio-temporal fluid dynamics36.&#160;Three-dimensional coherent structures which form downstream of the oscillating vocal folds have been studied by Neubauer et al.37; vortex generation and convection and jet flapping was observed. Recently, Krebs et al.38 studied the three-dimensionality of the glottal jet using stereoscopic PIV and the results demonstrate glottal jet axis switching. Erath and Plesniak14 investigated the effect of a model vocal fold polyp on the medial surface of a 7.5 times scaled-up dynamically driven vocal fold model. A recirculation region was formed downstream of the polyp and the jet dynamics were affected throughout the phonatory cycle. The previous studies, barring the driven vocal fold polyp study by Erath and Plesniak14, have not explored the fluid dynamics induced by a medial vocal fold polyp or nodule.
It is important to understand the fluid dynamic effect of the model polyp within steady and pulsatile flow fields before including the additional complexity of the vocal fold moving walls, induced pressure gradients, confined geometric volume and other intricacies. The current work focuses on the signature of the flow structures on the downstream wall under both steady and unsteady flow conditions. The interactions between the vortical structures that are shed from a protrusion and the downstream wall is of great interest for the investigation of vocal fold polyps as well as other biological considerations, as these interactions elicit a biological response.

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			<td style="width:227px;">0.063&#34; Diameter and 1&#34; Length</td>
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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.

Previous work using a 7.5 times scaled-up dynamically driven vocal fold model has demonstrated that the presence of a geometric protuberance, model vocal fold polyp, disrupts the normal dynamics of the glottal jet throughout the phonatory cycle. Representative results from the previous driven vocal fold model study are displayed in Figure 2 and Video 2. The video demonstrates the motion of the driven vocal folds as they change from a convergent to a divergent geometry. The vocal fold mod...

Watch this Scientific Journal Video about Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp at JoVE.com

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

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. ...

investigating-three-dimensional-flow-separation-induced-model-vocal

Research

JoVE Journal

Bioengineering

8.4K Views.  The George Washington University. Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient'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.

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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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 ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...