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

Abstract

Introduction Voice rest following phonotrauma or phonosurgery has a considerable clinical impact, but clinical recommendations are inconsistent due to inconclusive data. As biopsies of the vocal folds (VF) for molecular biology studies in humans are unethical, we established a new in vitro model to explore the effects of vibration on human vocal fold fibroblasts (hVFF) in an inflammatory and normal state, which is based on previously published models. Methods By using a phonomimetic bioreactor we were able to apply predefined vibrational stress patterns on hVFF cultured under inflammatory or normal conditions. Inflammatory and pro-fibrotic stimuli were induced by interleukin (IL)1β and transforming growth factor (TGF)β1, respectively. Mechanical stimulation was applied four hours daily, over a period of 72 hours. Outcome measurements comprised assessment of extracellular matrix (ECM)-related components, angiogenic factors, and inflammatory and fibrogenic markers on gene expression and protein levels. Results Under inflammatory conditions, the inflammatory cytokine IL11, as well as the myofibroblast marker alpha smooth muscle actin (α-SMA) were significantly reduced when additional vibration was applied. The desirable anti-fibrotic ECM component hyaluronic acid was increased following cytokine treatment, but was not diminished following vibration. Conclusion Our experiments revealed the effect of vibrational stress on hVFF in an inflammatory state. Elevated levels of certain pro-inflammatory/pro-fibrotic factors could be mitigated by additional vibrational excitation in an in vitro setting. These findings corroborate clinical studies which recommend early voice activation following an acute event.

Molekulare Laryngologie

“…Dass von diesen Zellen u. a. mehr Kollagen und weniger Hyaluronsäure produziert wird, ist das morphologische Korrelat der SL-Narbe [ 27 ]. Ergebnisse der In-vitro-Versuche der Autor(inn)en zeigten, dass eine unmittelbare mechanische Aktivierung nach inflammatorischem Stimulus zu einer niedrigeren Expression inflammatorischer und fibrotischer Gene führten [ 28 ]. Auch Geschlechtsunterschiede in der ECM-Komposition stehen im Verdacht, Auswirkungen auf die Häufigkeit von SL-Veränderungen zu haben: Die Lamina propria von Männern weist mehr Hyaluronsäure als die von Frauen auf, wodurch ein stärkerer Schutz vor Phonotraumen ausgehen könnte [ 29 ].…”

“…Die Arbeitsgruppe der Autor(inn)en hat in den letzten Jahren Protokolle entwickelt, mit welchen sie Epithelzellen und Fibroblasten aus laryngealer Mukosa isolieren, charakterisieren und kultivieren kann [ 28 , 33 , 41 ]. Ein weiterer wesentlicher Fortschritt war die Entwicklung geeigneter Zellkulturapparate, die die SL-Vibrationen auch unter Kulturbedingungen applizieren können.…”

“…Weltweit gibt es etwa 6 verschiedene Prototypen für derartige „Bioreaktoren“, wobei keiner davon kommerziell erhältlich ist. Die Gruppe der Autor(inn)en hat in den letzten Jahren einen derartigen „phonomimetischen“ Bioreaktor entwickelt und führt damit bereits Experimente zu verschiedenen Fragstellungen durch [ 28 , 34 , 41 ]. Der Vorteil des Prototypen der Autor(inn)en besteht darin, dass er aus kommerziell erhältlichen Einzelteilen besteht und deswegen relativ leicht von anderen Gruppen nachgebaut werden kann.…”

Influence of 40 Hz and 100 Hz Vibration on SH-SY5Y Cells Growth and Differentiation—A Preliminary Study

“…Vibrations also apply in regenerative medicine in bioreactors that mimic the natural environment for cell culture or stimulate cell differentiation [ 14 , 15 , 48 , 49 ]. Previous research findings showed that vibrations could affect stem cell behaviors and lead to the production of mature tissue [ 6 ].…”

In Vitro Evaluation of Biomaterials for Vocal Fold Injection: A Systematic Review

“…Therefore, unregulated inflammatory response is shown to be an adverse event in the vocal fold. The molecules involved in the inflammatory response in the vocal fold have been extensively studied for at least a decade [ 23 , 24 ]. The consistency of studies in quantifying IL-1β, NF-ĸB, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), transforming growth factor-beta (TGF-β), cyclooxygenase (COX2), HA and procollagen expression indicated that these molecules are closely related to inflammation in the vocal fold [ 25 ].…”