Development and validation of a novel phonomimetic bioreactor

Abstract

Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold’s lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFβ-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device.

In vitro mechanical vibration down-regulates pro-inflammatory and pro-fibrotic signaling in human vocal fold fibroblasts

“…They concluded that dynamic stimulation at appropriate amplitudes might have an anti-inflammatory effect, which is in accordance with our results. However, their device could only apply a very limited range of frequencies, whereas our device covers the entire range of the human voice [ 29 ].…”

“…Importantly, vibration did not diminish HA levels, which is significant since this glycosaminoglycan has important anti-fibrotic properties. Some other studies on the contrary showed changes in the HA metabolism following mechanical stimulation [ 25 , 29 ]. This might be due to differences in stimulatory patterns and their duration, since it is known that VF tissue responds differently depending on the type of phonatory stimulus [ 66 ].…”

“…The gene expression level of ACTA2 , the gene encoding for α-SMA, also decreased, but without reaching statistical significance. It is known that VFF transformation into myofibroblasts during inflammation is accompanied by an increase in α-SMA, a well-established myofibroblast marker [ 29 , 69 , 70 ]. Myofibroblasts appear to be responsible for wound contraction as well as increased collagen deposition and could lead to a deterioration of VF oscillation [ 70 ].…”

“…Mechanical stimulation was performed using a phonomimetic bioreactor developed and established by our group. In short, the flexible bottomed 6-well plates were oscillated by aerodynamic pressure fluctuation induced by a loudspeaker beneath the plate [ 29 , 49 ].…”

“…Our recently designed phonomimetic bioreactor allowed us to apply specific vibrational patterns to hVFF in culture [ 29 ]. Using this device, we were able to study cellular responses of hVFF following a pro-fibrotic and inflammatory stimulus under static and dynamic conditions for the first time.…”