Successful development of new therapeutic regenerative interventions for vocal fold disease, in addition to an improved understanding of molecular development, pathogenesis and biological features of the vocal fold lamina propria extracellular matrix (ECM) depend on the availability of reproducible in vitro
models. Bioreactors are laboratory tissue culture devices that provide a controllable, mechanically active environment that can be used to study and potentially improve engineered tissue structure, properties and integration. The use of bioreactors has been successful in regeneration of bone, vascular and cardiac tissue 
. The development and optimization of a unique bioreactor can be utilized to address the fundamental question of how mechanical modulation affects cell behavior and its production of ECM specific to the vocal fold. This knowledge will allow for the development of an ideal engineered cell seeded vocal fold construct.
Cell sourcing in the development of a biomimetic construct for the vocal fold is problematic. A distinct cell source of healthy native human hVFF does not exist. Normal vocal fold tissue from live donors is virtually impossible to obtain, and when they are obtained, insufficient numbers of cells are more often then not cultivated. Six immortalized hVFF lines have been developed, banked and have been used for in vitro investigation 
; immortalized lines are not an ideal universal donor line. Rather to be successful there needs to be the availability of distinct cell sources for tissue regeneration, cell characterization and development of universal donor cell lines 
. BM-MSC have been utilized in other areas of tissue engineering 
and given their regenerative potential and similarity to hVFF, comparisons between their behaviors in a developed bioreactor were made.
BM-MSC and hVFF had similar gene expression results such that vibration did not induce up or down regulation of collagen 1 or Fn. This is important as it shows that extended vibration may not cause hVFF to build up the ECM, creating a more fibrotic vocal fold mucosa. The lack of upregulation of these ECM proteins in BM-MSC is also important, as it indicates that they could potentially be placed within vocal fold tissue without contributing to fibrosis. Our results do differ from previously published vibration reports that have subjected laryngeal fibroblasts to strain and vibration 
. This discrepancy could occur as the result of several phenomena. hVFF were assessed in the present study, whereas laryngeal fibroblasts, more specifically tracheal scar fibroblasts have been used in other reports. It is likely that fibroblasts from different parts of the body respond differently to the same stimuli as fibroblasts from distinct anatomical sites show variable levels of expression from large groups of expressed genes 
. Vibration frequency and duration is also markedly different between studies. Lastly, other versions of vibrational bioreactors only vibrate one strip whereas the current bioreactor vibrates two strips that contact each other as a result of the sinusoidal frequency. It is likely that one or a combination of these differences caused the observed difference in results.
Evidence of SMA expression was not observed in any cell type indicating that neither BM-MSC nor hVFF display a myofibroblast phenotype following vibration 
. Myofibroblasts are present in nearly all fibrotic states, and are an important part of the wound healing response. The fact that these cells do not exhibit myofibroblast characteristics would seem to indicate that 8 hours of constant vibration does not elicit a strong wound healing response. Previous vocal fold bioreactor studies have not examined TGF-β1 expression levels due to strain or vibration. Our results show that though hVFF and BM-MSC both constitutively express mRNA for TGF-β1, there is no evidence that vibration increases expression in either cell type. This finding is also consistent with CIα1 findings, as increased levels of TGF-β1 has been shown to increase collagen secretion 
. TGF-β1 treatment is also associated with differentiation towards a myofibroblast phenotype in hVFF. The lack of myofibroblast markers further supports this finding.
Differences were found in regards to proliferation and apoptosis between the two cell types. As indicated by the TUNEL assay, high cell viability (96%) was maintained for both hVFF and BM-MSC, yet BM-MSC had significantly more apoptotic cells. It should be noted that less than 4% of BM-MSC in the vibrated condition underwent apoptosis after 8 hours of constant vibration. This difference may not be of strong impact as 8 hours of constant vibration is far longer than is typical for heavy voice users 
. Our immunohistochemistry data indicate that the sustained vibration condition leads to an increase of fibroblast proliferation. Previous studies have found that externally applied static strain can induce proliferation in a variety of cells, including fibroblasts 
. In these pathways, the MAPK/ERK pathway is activated due to receptor stimulation at focal adhesions. In particular, ERK2 has shown to be activated simply due to stretch on fibronectin 
. Given this evidence, there is potential that this pathway was activated in the vibrated hVFF. Further study is needed to further discern the effects of applied vibration and strain.
Several limitations of our investigation warrant discussion. For the hVFF, an immortalized cell line was used instead of primary cells. It is possible that the immortalized cells would respond differently than primary cells, although previous work indicates that the response should be similar 
. Only one cell line for each cell type was used, and as such each cell type only came from one respective donor. The limited number of donors does not allow the data to show if the results differ between individuals. The differences in BM-MSC response between donors for other fields is documented 
, and it stands to reason that some difference may be seen here as well. In addition, only one vibration pattern was used (i.e, continuous vibration for 8 hours) in the present study. In order to more accurately quantify the effects of vibration, more than one paradigm needs to be used. A pattern more consistent with typical speech, such as vibrate for 30 seconds, rest for 10 seconds, repeated for 8 hours may provide insight into the effects of a normal day of speaking on these cell types. The frequency, 200 Hz, is also closer to the female voice range as compared to the male range. It is possible that a different response may have been seen at a lower frequency, such as 110 Hz, closer to a typical male speaking voice. Finally, a limited number of genes were investigate in this study. Though no difference was seen following vibration with the chosen ECM genes, the expression of other genes may have changed. The number of gene targets should be expanded to further determine the changes, or lack thereof, following vibration.
A newly developed vibration bioreactor, capable of causing two cell seeding substrates to contact each other in a wave-dependent motion, was designed and tested. Mechanical forces applied to BM-MSC and hVFF did not yield significant gene expression differences after 8 hours of vibration. BM-MSC may be an ideal alternate for the development of a bimimetic tissue engineered construct for vocal folds. Future studies will investigate the effects of vibration using more donors, while also investigating more protein and gene markers.