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....

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 b....

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 waveforms with desire.......

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

List of materials used in this article

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 O/N
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 (80-13,000 Hz), 85 dB
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