We thank Dr. Jeffrey Caplan for his training and advice on confocal imaging. We also thank the Keck Electron Microscopy Lab and Dr. Chaoying Ni for SEM assistance. This work is funded by National Institutes of Health (NIDCD, R01DC008965 and R01DC011377). ABZ acknowledges NSF Integrative Graduate Education &#38; Research Traineeship (IGERT) program for funding.

1. Bioreactor Assembly (Video 1)
<ol>
	<li>Make an aluminum mold (circular die + spacer pin) with pre-determined inner and outer dimensions (Figure&#160;1).</li>
	<li>Using the mold from step 1.1, fabricate a silicone membrane (diameter: 42 mm, thickness: 1.5 mm, Figure&#160;1) with an entrenched sleeve (diameter: 12 mm, thickness ~0.25 mm, shaped by the spacer pin in Figure 1) in the middle using a commercially available silicone elastomer kit.</li>
	<li>Make a pair of acrylic blocks (Figure&#160;2 (4, 5)) with a circular opening (diameter: 24 mm) in the middle, engrave the top (1.8 cm thick) and bottom (0.9 cm thick) blocks with matching ridges and grooves.10</li>
	<li>Sandwich the silicone membrane between the paired acrylic blocks. Secure the assembly with four corner screws using a micro torque screwdriver set to a constant force (35 cN&#183;m). As a result, a water-tight, 24 mm wide and 18 mm deep vibration chamber is created (Figure&#160;2C).</li>
	<li>Mount a 3&#8221; extended range mini-woofer (Figure&#160;2D, 8 &#937;/20 W) underneath the vibration chamber through another set of corner screws on the bottom acrylic block. At this point, an individual vibration module is assembled.</li>
	<li>Replicate seven additional vibration modules. Affix four of them to one of two stationary aluminum bars (40 cm x 10 cm x 2.5 cm) by placing the speaker bases in evenly spaced circular holes (diameter: 7 cm, thickness: 2 cm) cut into the bars. Stabilize each speaker by inserting a screw through the side of the aluminum bar into each circular hole.</li>
	<li>Individually control the speakers by a speaker selector. Connect individual speakers to the selector by attaching wires to the positive and negative inputs on the speaker body then to the corresponding outputs on the selector. The speaker selector allows the signal from the function generator, after passing through a power amplifier, to reach all eight speakers at once (Figure&#160;2E).</li>
	<li>Place the two chamber arrays, the speaker selector and associated electronics in an anti-humidity enclosure. House the entire assembly in a commercial cell culture incubator.</li>
	<li>Feed the main cables (through a medical grade PVC tubing) connecting the power amplifier and speaker selector through the filter assembly at the back of the incubator.</li>
</ol>
2. Scaffold Fabrication and Characterization
<ol>
	<li>Dissolve PCL pellets in chloroform at a concentration of 15 wt%. Load the solution into a 10 ml syringe capped with a 21 G blunt-ended needle.</li>
	<li>Lock the syringe onto a programmable syringe pump and set the flow rate at 1 ml/hr.</li>
	<li>Place the aluminum foil-covered collector across from the needle horizontally, with a needle tip-to-collector distance of ~18 cm.</li>
	<li>Clamp the positive alligator clip to the middle of the needle, and the ground alligator clip to the aluminum collector, then set the voltage on the high voltage power supply at 15 kV. CAUTION: high voltage, keep distance from the needle.</li>
	<li>Sequentially turn on the syringe pump and power supply; quickly clean/remove the residual polymer solution surrounding the tip of the needle using a dry paper towel before stable fiber jets and Taylor cone19 are formed.</li>
	<li>Allow the fibers to accumulate on the Al collector to a thickness of ~250-300 &#181;m (~7 hr under the current spinning conditions). Store the resultant scaffolds in a vacuum desiccator for 1-2 days to remove any residual solvent.</li>
	<li>Image the scaffolds, sputter coated with gold, using a Scanning Electron Microscope to show consistent fiber morphology.10</li>
</ol>
3. Bioreactor Assembly and Characterization
<ol>
	<li>Punch a cylindrical disk (diameter: 8 mm) with four arms (length: 2 mm) out of the as-spun PCL mat (Figure&#160;2A) by first using a 12 mm diameter biopsy punch to cut the outer diameter of the disk. Then use a second, 8 mm biopsy punch to make four 2 mm long notches evenly spaced around the circular blade to score where the arms are to be cut. After scoring with the 8 mm punch, use a scalpel blade to cut the edges of the arms outward. Insert the scaffold into the groove of the silicone membrane via the extended arms (Video 1). Flatten the inserted scaffold by gently pressing the surface using flathead tweezers.</li>
	<li>Attach a small piece of thin Al foil (8 mm x 2 mm, orthogonal shape, Figure&#160;2B) to the PCL scaffold to aid laser reflection.</li>
	<li>Secure the assembled silicone membrane/PCL scaffold (as detailed in step 1.4) in the vibration chamber. Add 1.5 ml water in the chamber in order to hydrate the PCL scaffold before vibration.</li>
	<li>Using the function generator, introduce vibration signals (e.g., 200 Hz sinusoidal waves with a peak-to-peak voltage, Vpp, of 0.1 V) to the sandwiched acrylic chamber. Use a voltmeter to accurately measure the voltage at each speaker input. Note: the Vpp readout from the function generator will differ from the eventual voltage delivered to the speaker.</li>
	<li>Assemble the single-point LDV and secure the fiber-optic laser sensor head to a pan-tilt head tripod. Angle the sensor head so that it is pointing perpendicular to the tabletop. Connect the LDV sensor head to the data acquisition module via coaxial cable then the module to the laptop via USB.</li>
	<li>Focus the laser beam perpendicularly at various predetermined points on the silicone membrane (Figure&#160;2B and Figure&#160;3).</li>
	<li>Using the data acquisition software, record the mid-membrane displacement. Click &#8220;Acquisition Settings&#8221; from the &#8220;Options&#8221; menu; then change the measurement mode to &#8220;FFT&#8221;. Next, click the &#8220;Continuous Measurement&#8221; in the main toolbar then click the peak that forms at the chosen frequency (Figure&#160;6D) to record displacement.</li>
	<li>Plot the normal mid-membrane displacement (w0) as a function of the relative position across the substrate. Construct a 3D colormap of the surface vibratory profile using Origin 8.5 data analysis software.</li>
</ol>
4. Vibratory Cell Culture
<ol>
	<li>Sub-culture human bone marrow-derived MSCs in T150 tissue culture flasks at an initial seeding density of 4,000-5,000 cells/cm2 in MSC maintenance media.</li>
	<li>After 7-8 days of cell culture (to ~85% confluency), trypsinize the cells with a cell dissociation reagent such as accutase, count using a hemocytometer, centrifuge (440 x g for 5 min), and re-suspend the cell pellet in fresh MSC maintenance media at a concentration of 4.5 x 106 cells/ml.</li>
	<li>Immerse the PCL scaffold in 70% ethanol O/N. After the solvent is evaporated, expose both sides of the scaffold to germicidal UV light (254 nm) for 5-8 min.</li>
	<li>Soak the PCL scaffold in a 20 &#181;g/ml fibronectin solution at 37 &#176;C for 1 hr. Insert the fibronectin-coated scaffold into the silicone membrane. Assemble the bioreactor as detailed in step 1.</li>
	<li>Distribute 40 &#181;l of the cell suspension evenly on the secured PCL scaffold. Allow the cells to attach for 1-1.5 hr before adding an additional 1.5 ml fresh media to the vibration chamber.</li>
	<li>Culture the MSC-laden PCL scaffold statically for 3 days and refresh the media upon completion of the static culture.</li>
	<li>Impose selected vibration regimes to the cellular constructs. Note: As an example, cells are subjected to a 1-hr-on-1-hr-off (OF) vibration at 200 Hz with a w0 of ~40 &#956;m for 12 hr per day for up to 7 days. Constructs subjected to vibratory stimulations are designated as Vib samples and those cultured statically in identical vibration chambers serve as static controls (Stat).</li>
</ol>
5. Biological Evaluations
<ol>
	<li>Collect 200 &#181;l cell culture media from each chamber every other day (day 1, 3, 5, and 7) and pool the aliquots from the same sample together (800 &#181;l each).</li>
	<li>Quantify the cellular production of matrix metalloproteinase-1 (MMP1) and HA using an MMP1 ELISA Development kit and a hyaluronan competitive ELISA kit, respectively, following the manufacturers&#8217; procedure. Meanwhile, assay the production of soluble elastin precursor following the previously reported ELISA procedure.8</li>
	<li>Upon completion of the last vibration cycle at day 7, quickly remove the cellular constructs from the vibration chambers using sharp tweezers and briefly rinse them with cold phosphate buffered saline (PBS, 4 &#176;C).</li>
	<li>For the live/dead staining, incubate the constructs with propidium iodide (1:2,000 in PBS) and Syto-13 (1:1,000 in PBS) simultaneously for 5 min at RT. Image the stained constructs with a multiphoton confocal microscope.</li>
	<li>Separately, snap-freeze the PBS-rinsed cellular constructs on dry ice and extract the total cellular RNA following a previously reported protocol for gene analysis.9</li>
	<li>Verify the quantity and quality of the extracted RNA using a UV-Vis spectrophotometer. RNA samples with A260/A280 and A260/A230 ratios of 1.8-2.2 are used for subsequent qPCR analysis.</li>
	<li>Reverse transcribe the RNA (500 ng/sample) into cDNA using a commercially available reverse transcription kit.</li>
	<li>Perform the PCR reaction on a real-time sequence detection system using a commercially available PCR master mix following the previously detailed procedure.8</li>
	<li>Analyze the qPCR results using commercial qPCR data analysis software. To ensure the reliability of the data analysis, multiple reference genes (YWHAZ, TBP, PPIA) are employed as internal controls, and the variance of specific primer efficiencies is taken into account.8</li>
</ol>

<ol>
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Ser. 1, 167-173 (2004).</li><li>Brown, W. S., Morris, R. J., Hollien, H., Howell, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speaking+Fundamental-Frequency+Characteristics+as+a+Function+of+Age+and+Professional.">Speaking Fundamental-Frequency Characteristics as a Function of Age and Professional.</a> J. Voice. 5, 310-315 (1991).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanotransduction:+putting+all+the+pieces+together+again.">Cellular mechanotransduction: putting all the pieces together again.</a> Faseb J. 20, 811-827 (2006).</li><li>Titze, I. 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No competing financial interests exist.

Successful engineering of functional vocal fold tissues in vitro requires the recreation of a vocal fold-like microenvironment to mediate the behaviors of multipotent cells. It is generally accepted that tissue or organ structures reflect the functions they are required to perform.22 For vocal fold tissues, the high frequency vibrations that occur during phonation are proposed to be important for tissue maturation. In our study, PCL scaffolds are used to provide a ligament-like structural support whil...

The human vocal fold, composed of an epithelial layer, the lamina propria (LP) and the vocalis muscle, is a specialized soft tissue that converts air flow from the lungs into acoustic waves for sound production.1 Vocal folds oscillate regularly during normal phonation, exhibiting strains of up to 30% at fundamental frequencies ranging from 100-300 Hz.2 Adult vocal fold LP is a gradient structure composed of a superficial (SLP), an intermediate (ILP) and a deep (DLP) layer. Further classification groups the epithelium and the SLP as the mucosa layer, and combines the ILP and DLP into the vocal ligament.3 The SLP layer contains primarily an amorphous matrix with sparsely dispersed collagenous fibers, while the ligament is enriched with mature collagen and elastin fibers to provide sufficient strength.4 The structure and mechanics of newborn vocal folds vary significantly from their mature counterparts. Although mechanisms regulating vocal fold development and maturation are not yet fully understood, experimental evidence has pointed to the defining roles of vocalization-derived mechanical stress.
Several medical conditions, including voice abuse, infections, chemical irritants and surgical procedures, can damage the vocal fold. Vocal fold disorders affect an estimated 3-9% of the U.S. population. Current treatment methods for vocal fold disorders are limited5 and a stem cell-based tissue engineering approach has emerged as a promising strategy for restoring vocal fold function. Mesenchymal stem cells (MSCs) are a suitable alternative to the primary vocal fold fibroblasts for vocal fold tissue engineering.6-9 Stem cell fate specification and subsequent tissue development are mediated by the specific niche they reside in, of which the mechanical condition is a vital factor.10 Mechanical forces are essential regulators of tissue morphogenesis and homeostasis, especially for tissues that are routinely subjected to loading.11 From a tissue engineering perspective, it has been demonstrated that exposure to physiologically relevant mechanical stimulations promotes stem cell differentiation and tissue-specific matrix remodeling.12-15
Tissue culture bioreactors are designed to simulate the desired physiological environment for cell or tissue growth in vitro. For vocal fold tissue engineering, it is especially critical to recreate the mechanical environment of the phonating vocal folds. An ideal vocal fold bioreactor should effectively deliver vibratory cues to culture cells, allowing facile control over frequency, amplitude and duration of vibrations. Titze and coworkers devised a vocal fold bioreactor (T1 bioreactor)16 that combines static stretch with high frequency (20-200 Hz) oscillations to stimulate the cellular production of matrix proteins. Using this bioreactor, Webb and colleagues17 studied the effects of 10-day, 100-Hz vibrations on dermal fibroblasts cultured in a hyaluronic acid (HA)-based hydrogel. Constructs subjected to vibration exhibited an elevated expression of HA synthase-2 (HAS2), decorin, fibromodulin and matrix metalloproteinase-1 (MMP1), relative to the static controls. The stimulatory effects were found to be time dependent. More recently, our group18 assembled a vocal fold bioreactor (J1 bioreactor) using a power amplifier, a function generator, an enclosed loud speaker and a circumferentially-anchored silicone membrane that transfers the oscillating air to the attached cells. Neonatal foreskin fibroblasts cultivated in the J1 bioreactor were subjected to 1 hr of vibration at 60, 110 or 300 Hz, with an in-plane strain of up to 0.05%. The qPCR results suggested that the expression of some ECM genes was moderately altered in response to the varied vibratory frequencies and amplitudes.
These bioreactor designs, while intriguing, have several limitations. For example, the T1 system requires a large number of connectors and bars for mechanical coupling, limiting the maximum frequencies attainable. Moreover, cells may be subjected to undesirable mechanical agitation and fluid perturbation that complicate the data interpretation. The J1 bioreactor, on the other hand, exhibits relatively low energy conversion efficiency and is not user-friendly. In addition, vibration frequently detaches the cell-laden constructs from the underlying silicone membrane. The J2 vocal fold bioreactor reported here, designed based on the same principle as the J1 version, is optimized for consistency and reproducibility. The phonation-mimicking vibrations are generated aerodynamically in individually fitted vibration chambers where MSC-populated fibrous poly(&#949;-caprolactone) (PCL) scaffolds are effectively secured. Laser Doppler Vibrometry (LDV) allows the user to verify the vibratory profile of the membrane/scaffold assembly. In our demonstration, MSCs are exposed to 200-Hz sinusoidal vibrations with a 1-hr-on-1-hr-off (OF) pattern for a total of 12 hr daily for 7 days. Cellular responses to the imposed vibratory cues are investigated systematically. Overall, the J2 vocal fold provides the most user-friendly features, allowing dynamic cell culture studies to be conducted in a high throughput and reproducible fashion.

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="16">
			<td height="16" style="height:17px;width:248px;">silicone elastomer kit</td>
			<td style="width:188px;">Dow Corning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td style="width:293px;">cure the membrane at 100 C for 2 hr</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCL</td>
			<td>Sigma Aldrich</td>
			<td>440744-500G</td>
			<td>Mn ~ 80 kDa, dissolve overnight</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">human bone marrow-derived MSCs</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>received with passage 2</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">MSC maintenance media</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td style="width:293px;">10% FBS in basal media supplemented 
			with L-glutamine, gentamicin and amphotericin</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-1MG</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">MMP1 DuoSet ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY901</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HA ELISA kit</td>
			<td>Echelon Biosciences</td>
			<td>&#160;K-1200&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">propidium iodide&#160;</td>
			<td>Life Technologies</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Syto-13&#160;</td>
			<td>Life Technologies</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">QuantiTect reverse transcription kit&#160;</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">SYBR Green PCR master mix</td>
			<td>Life Technologies</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">replacement speaker</td>
			<td style="width:188px;">DAYTON audio 
			(via Parts Express)</td>
			<td>DS90-8</td>
			<td>paper cone, full range (80-13000 Hz), 85dB</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Ergo Micro torque screwdriver</td>
			<td>Mountz</td>
			<td># 020377</td>
			<td>torque range: 20-120 cN.m</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">stereo speaker selector</td>
			<td>RadioShack</td>
			<td>40-244</td>
			<td>maximum power handling 50 W</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">function generator&#160;</td>
			<td>Agilent&#160;</td>
			<td>33220A</td>
			<td>frequency range 1 &#181;Hz- 20 MHz</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">power amplifier&#160;</td>
			<td>PYLE audio</td>
			<td>PylePro PT2400</td>
			<td style="width:293px;">frequency response: 10 Hz-50 kHz, two speaker 
			channels</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">cell culture incubator&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Steri-Cult 3307</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">syringe pump&#160;</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE 1000R</td>
			<td>output voltage: 0-30 kV</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">scanning electron microscope&#160;</td>
			<td>JEOL-USA</td>
			<td>JSM-7400F</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">desk gold sputter coater</td>
			<td>Denton Vacuum</td>
			<td>DSK00V-0013</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Doppler laser vibrometer&#160;</td>
			<td>Polytec</td>
			<td>PDV-100</td>
			<td>non-contact velocity measurement (0-22 kHz)</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCR sequence detection system&#160;</td>
			<td>Applied Biosystems</td>
			<td>ABI7300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">multiphoton confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510Meta NLO</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV-VIS Spectrophotometer&#160;</td>
			<td style="width:188px;">NanoDrop Products 
			via Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">VibSoft Data Acquisition Software</td>
			<td>Polytec</td>
			<td></td>
			<td>acquisition bandwidth up to 40 MHz</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Origin 8.5 data analysis software&#160;</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">qbasePlus qPCR data analysis software&#160;</td>
			<td>Biogazelle</td>
			<td>V2.3</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">aluminium alloy&#160;</td>
			<td>McMaster-Carr</td>
			<td>Alloy 6061</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">acrylic blocks</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">polycarbonate anti-humidity chamber</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td style="width:293px;">Impact-Resistant&#160;Polycarbonate</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">screws&#160;</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">electronic cable/wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">medical grade PVC tubing</td>
			<td>US Plastic Corp.</td>
			<td>Tygon S-50-HL</td>
			<td>clear, biocompatible</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">10 mL syringe&#160;</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">21 G blunt ended needle</td>
			<td>Small Parts</td>
			<td>NE-213PL-25</td>
			<td>1-1/2&#34; length</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Alligator clip adapters&#160;</td>
			<td>RadioShack</td>
			<td>270-354</td>
			<td>fully insulated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">8 mm biopsy punch</td>
			<td><a href="http://www.sklarcorp.com/ProductListUS.aspx?DepartmentID=10&amp;CategoryID=96">Sklar&#160;Surgical Instruments</a></td>
			<td>96-1152</td>
			<td>sterile, disposable</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">12 mm biopsy punch</td>
			<td>Acuderm (via Fisher Scientific)</td>
			<td style="width:124px;">NC9998681</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">tissue culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>cell culture treated</td>
		</tr>
	</tbody>
</table>

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.

The PCL scaffolds fabricated by electrospinning contain micron sized interstitial pores and randomly entangled fibers with an average diameter of 4.7 &#181;m (Figure&#160;4A). At a higher magnification, nanoscale grooves and pores are visible on individual fibers (Figure&#160;4B). Coating of the scaffolds with fibronectin improves hydrophilicity and facilitates the initial cell adhesion/spreading on the PCL scaffold (unpublished observation).
Sinusoidal wavefo...

Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Construction and Characterization of a Novel Vocal Fold Bioreactor

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 ...

construction-and-characterization-of-a-novel-vocal-fold-bioreactor

Research

JoVE Journal

Bioengineering

9.1K Views.  University of Delaware. 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(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Video: Construction and Characterization of a Novel Vocal Fold Bioreactor

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

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 ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is 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. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...