Abstract
Illumination of cellular changes caused by mechanical forces present within the laryngeal microenvironment may well guide strategies for tissue engineering the vocal fold lamina propria. The purpose of this study was to compare the response of human vocal fold fibroblasts (hVFF) and bone marrow mesenchymal stem cells (BM-MSC) to vibratory stimulus. In order to study these effects, a bioreactor capable of vibrating two cell seeded substrates was developed. The cell seeded substrates contact each other as a result of the sinusoidal frequency, producing a motion similar to the movement of true vocal folds. Utilizing this bioreactor, hVFF and BM-MSC were subjected to 200 Hz vibration and 20% strain for 8 hours. Immunohistochemistry (Ki-67 and TUNEL) was performed to examine cell proliferation and apoptosis respectively, while semi-quantitative RT-PCR was used to assess extracellular matrix related gene expression. HVFF significantly proliferated (p = 0.011) when subjected to 200 Hz vibration and 20% strain, while BM-MSC did not (p = 1.0). A statistically significant increase in apoptosis of BM-MSC (p = 0.0402) was observed under the experimental conditions; however high cell viability (96%) was maintained. HVFF did not have significantly altered apoptosis (p = 0.7849) when subjected to vibration and strain. Semi-quantitative RT-PCR results show no significant differences in expression levels of collagen I (BM-MSC p = 0.1951, hVFF p = v0.3629), fibronectin (BM-MSC p = 0.1951, hVFF p = 0.2513), and TGF-β1 (BM-MSC p = 0.2534, hVFF p = 0.6029) between vibratory and static conditions in either cell type. Finally, smooth muscle actin mRNA was not present in either vibrated or static samples, indicating that no myofibroblast differentiation occurred for either cell type. Together, these results demonstrate that BM-MSC may be a suitable alternative to hVFF for vocal fold tissue engineering. Further investigation into a larger number of gene markers, protein levels, increased number of donors and vibratory conditions are warranted.
Highlights
The unique mechano-environment of vocal fold mucosa includes its ability to sustain oscillation up to 1000 hertz, amplitudes of 1 mm, and accelerations of 200–300 G [1,2]
Recent work by Wolchok et al support this finding by showing that laryngeal fibroblasts upregulate ECM related mRNA, and secrete matrix proteins, including collagen I and fibronectin, when exposed to mechanical vibration [11]. It is unknown if human vocal fold fibroblasts, the cells that are responsible for the production of the extracellular matrix of the vocal fold mucosa [12], respond to vibration in a similar fashion to remodel the ECM as fibroblasts from other locations in the larynx
Bioreactor Design Our current model of bioreactor simulates the vocal fold in vivo environment by subjecting cell-seeded synthetic extracellular matrix strips to three stimuli – vibration, tensile stress, and dynamic angle change. sECM strips are mounted in a replaceable, sterile T-150 cell culture flask, which has an open upper surface, fastened to the bioreactor base (Figure 1)
Summary
The unique mechano-environment of vocal fold mucosa includes its ability to sustain oscillation up to 1000 hertz, amplitudes of 1 mm, and accelerations of 200–300 G [1,2]. Recent work by Wolchok et al support this finding by showing that laryngeal fibroblasts upregulate ECM related mRNA, and secrete matrix proteins, including collagen I and fibronectin, when exposed to mechanical vibration [11]. It is unknown if human vocal fold fibroblasts (hVFF), the cells that are responsible for the production of the extracellular matrix of the vocal fold mucosa [12], respond to vibration in a similar fashion to remodel the ECM as fibroblasts from other locations in the larynx. An understanding of how hVFF alter ECM production and degradation in response to vibration is crucial for learning how to direct tissue growth in a future bioengineered vocal fold mucosa replacement
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