Construction and Characterization of a Novel Vocal Fold Bioreactor

Summary

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.

Abstract

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 µm. 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.

Introduction

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(ε-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.

Protocol

1. Bioreactor Assembly (Video 1)

1. Make an aluminum mold (circular die + spacer pin) with pre-determined inner and outer dimensions (Figure 1).
2. Using the mold from step 1.1, fabricate a silicone membrane (diameter: 42 mm, thickness: 1.5 mm, Figure 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.
3. Make a pair of acrylic blocks (Figure 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
4. 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·m). As a result, a water-tight, 24 mm wide and 18 mm deep vibration chamber is created (Figure 2C).
5. Mount a 3^” extended range mini-woofer (Figure 2D, 8 Ω/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.
6. 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.
7. 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 2E).
8. 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.
9. 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.

2. Scaffold Fabrication and Characterization

1. 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.
2. Lock the syringe onto a programmable syringe pump and set the flow rate at 1 ml/hr.
3. Place the aluminum foil-covered collector across from the needle horizontally, with a needle tip-to-collector distance of ~18 cm.
4. 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.
5. 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.
6. Allow the fibers to accumulate on the Al collector to a thickness of ~250-300 µm (~7 hr under the current spinning conditions). Store the resultant scaffolds in a vacuum desiccator for 1-2 days to remove any residual solvent.
7. Image the scaffolds, sputter coated with gold, using a Scanning Electron Microscope to show consistent fiber morphology.¹⁰

3. Bioreactor Assembly and Characterization

1. Punch a cylindrical disk (diameter: 8 mm) with four arms (length: 2 mm) out of the as-spun PCL mat (Figure 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.
2. Attach a small piece of thin Al foil (8 mm x 2 mm, orthogonal shape, Figure 2B) to the PCL scaffold to aid laser reflection.
3. 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.
4. 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.
5. 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.
6. Focus the laser beam perpendicularly at various predetermined points on the silicone membrane (Figure 2B and Figure 3).
7. Using the data acquisition software, record the mid-membrane displacement. Click “Acquisition Settings” from the “Options” menu; then change the measurement mode to “FFT”. Next, click the “Continuous Measurement” in the main toolbar then click the peak that forms at the chosen frequency (Figure 6D) to record displacement.
8. 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.

4. Vibratory Cell Culture

1. 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.
2. 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.
3. 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.
4. Soak the PCL scaffold in a 20 µg/ml fibronectin solution at 37 °C for 1 hr. Insert the fibronectin-coated scaffold into the silicone membrane. Assemble the bioreactor as detailed in step 1.
5. Distribute 40 µl 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.
6. Culture the MSC-laden PCL scaffold statically for 3 days and refresh the media upon completion of the static culture.
7. 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 μm 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).

5. Biological Evaluations

1. Collect 200 µl 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 µl each).
2. 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’ procedure. Meanwhile, assay the production of soluble elastin precursor following the previously reported ELISA procedure.⁸
3. 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 °C).
4. 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.
5. 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
6. 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.
7. Reverse transcribe the RNA (500 ng/sample) into cDNA using a commercially available reverse transcription kit.
8. Perform the PCR reaction on a real-time sequence detection system using a commercially available PCR master mix following the previously detailed procedure.⁸
9. 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

Results

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

Sinusoidal wavefo...

Discussion

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...

Materials

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