Scalable and High-Throughput In Vitro Vibratory Platform for Vocal Fold Tissue Engineering Applications

Abstract

The vocal folds (VFs) are constantly exposed to mechanical stimulation leading to changes in biomechanical properties, structure, and composition. The development of long-term strategies for VF treatment depends on the characterization of related cells, biomaterials, or engineered tissues in a controlled mechanical environment. Our aim was to design, develop, and characterize a scalable and high-throughput platform that mimics the mechanical microenvironment of the VFs in vitro. The platform consists of a 24-well plate fitted with a flexible membrane atop a waveguide equipped with piezoelectric speakers which allows for cells to be exposed to various phonatory stimuli. The displacements of the flexible membrane were characterized via Laser Doppler Vibrometry (LDV). Human VF fibroblasts and mesenchymal stem cells were seeded, exposed to various vibratory regimes, and the expression of pro-fibrotic and pro-inflammatory genes was analyzed. Compared to current bioreactor designs, the platform developed in this study can incorporate commercial assay formats ranging from 6- to 96-well plates which represents a significant improvement in scalability. This platform is modular and allows for tunable frequency regimes.

Keywords: bioreactor; displacement; fibroblasts; frequency; gene expression; mesenchymal stem cells; piezoelectric speaker; vibration; vocal fold.

Figure 1

Overview. (A) Schematic of the in vitro vibratory platform in which a piezoelectric speaker is attached to a polydimethylsiloxane (PDMS) waveguide that allows the waves to be propagated to a cell culture plate. The speaker is connected to an amplifier which can be controlled via a computer to input the frequency and sound volume. (B) Timeline of experiments.

Figure 2

Platform fabrication. (A) Step 1: support frame and PDMS waveguide: (i) real-life image of the 3D printed support frame, (ii) a well plate lid is placed on the 3D printed frame to prevent leaking, (iii) a foam and tape wall is placed on the side of the frame, (iv) PDMS is poured and cured for 4 days. (B) Step 2: piezoelectric speaker placement: (i) a 3D printed frame is (ii) placed on the PDMS waveguide, and (iii) six piezoelectric speakers are placed equidistantly. (iv) Top view of the assembled PDMS waveguide with attached piezoelectric speakers. (C) Step 3: cell culture plate preparation: the Tegaderm^TM flexible membrane is attached to the bottom of a 24-well bottomless plate. (D) Overall assembly order of each component from Steps 1–3.

Figure 3

Fully assembled system placed on the benchtop.

Figure 4

Average axial Tegaderm^TM dressing displacement in μm with respect to speaker frequency and volume. (A) Computer generated schematic of bioreactor positioning on the LDV XY Precision Stage. (B) LDV measured displacement of Tegaderm^TM dressing in the Z-axis. (C) LDV measured displacement of Tegaderm^TM dressing in the X-axis. (D) LDV measured displacement of Tegaderm^TM dressing in the Y-axis. Bar graphs represent Box and Whiskers plots showing all measured points. * = statistically significant (p < 0.05).

Figure 5

(A) Heatmap of LDV measured 3D vector magnitude of Tegaderm^TM membrane displacement (µm) vibrated at 100 Hz and 100% sound volume. (B) Gene expression profiles for ACTA2, MMP1, and HAS1 for HVOX and hMSCs cultured on top of Tegaderm^TM membrane and vibrated at 100 Hz and 100% sound volume for 2 h. Low = 28–35 µm, mid = 42–52 µm, high = 83–92 µm. Bar graphs represent mean ± standard error of the mean (SEM). n = 3 samples analyzed per experimental group. * = statistically significant (p < 0.05). n.s. = not significant (p > 0.05).