The response of vocal fold fibroblasts and mesenchymal stromal cells to vibration

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.

Figure 1. Schematic of the developed bioreactor.

A. Bioreactor including T-flask, substrate, voice-coil actuator, linear stepper motors, rotary stepper motors, and scissor bars. B. Experimental setup, with static attachment in place of stepper motors. Non-vibrated controls can also be seen. C. Bioreactor within incubator. Wave-form generator is next to the incubator, sitting on top of the power amplifier.

Figure 2. Proliferation of hVFF and BM-MSC.

A. Representative 20× of vibrated and control immunohistochemistry for each cell type. Proliferating cells are marked by TRITC (red), cell nucleus (blue DAPI). B. Percentage of proliferating cells under each condition. An increase in proliferation is observed with vibrated hVFF compared to controls. *(p = .011).

Figure 3. Apoptosis in hVFF and BM-MSC.

A. Representative 20× pictures of vibrated and control immunohistochemistry sections for each cell type. Apoptotic cells are marked by FITC (green), cell nuclei DAPI (blue). B. The percentage of proliferating cells under each condition. An increase in apoptosis is observed with vibrated BM-MSC compared to controls. * (p = .0402).

Figure 4. Gene Expression for hVFF and BM-MSC.

A. Box and whisker plot showing expression changes for CIα1. No statistical difference was found between vibrated and control for either cell type. B. Box and whisker plot showing expression changes for FN. No statistical difference was measured between vibrated and control for either cell type. C. Box and whisker plot showing expression changes for TGF-β1. No statistical difference was measured between vibrated and control for either cell type.