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.
Highlights
The exploration of molecular pathways in vocal fold (VF) biology and novel treatment strategies is impeded by the lack of knowledge of cellular response to relevant mechanical stimuli, primarily vibration
The wells were filled with 2.5 ml of medium, a typical volume used in cell culture for further measurements
The excitation was elicited by sound waves, The mode shapes plotted were an average of all six membranes in one cell culture plate
Summary
The exploration of molecular pathways in vocal fold (VF) biology and novel treatment strategies (e.g. laryngeal tissue engineering) is impeded by the lack of knowledge of cellular response to relevant mechanical stimuli, primarily vibration. An intact LP is essential for physiological function and is mainly composed of vocal fold fibroblasts (VFF) and extracellular matrix (ECM) components (collagen fibres, hyaluronic acid etc.). It is divided in three distinct layers based on the histological composition of the ECM [2]. The resulting deterioration of the voice (dysphonia) reduces quality of life, leads to social withdrawal and affects the ability to work (e.g. teachers, call-center agents) [8]
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