In vitro studies suggest early voice activation as an appropriate treatment after the vocal folds damage

Early tissue repair is accompanied by an inflammatory reaction, which can prevent infection at the site of injury and promote wound healing. However, it also carries a risk of complications. When the vocal folds (VF) heals, it can at worst lead to persistent dysphonia due to scarring of the VF. That’s why it is so important to appropriately treat VF after an injury. However, clinical recommendations after phonotrauma (surgery or abnormal voice emission) are inconsistent. Some point to the voice unusing, others to its early activation, as having a significant clinical impact. Therefore, Austrian scientists from the Medical University of Graz decided to investigate the effect of early voice activation on post-phonotrauma treatment. For ethical reasons, they used a special in vitro research model for this purpose.

• After mechanical stimulation under inflammatory conditions, a significant decrease in proteins associated with fibrosis was observed:
• the concentration of IL11 decreased approx. 3 times
• and the level of α-SMA decreased approx. 3 times.

Prepared on the basis of:

In vitro mechanical vibration down-regulates pro-inflammatory and pro-fibrotic signaling in human vocal fold fibroblasts. Hortobagyi D, Grossmann T, Tschernitz M, Grill M et al.  PLoS One. 2020 Nov 19;15(11):e0241901.

Study population

Cell cultures of human vocal fold fibroblasts (hVFF).

Test procedure

Using a phonomimetic bioreactor, scientists were able to apply predefined vibrational stress patterns on hVFF, grown under normal or inflammatory conditions. Inflammatory and fibrogenic (promoting fibrosis and scar formation) stimuli were induced by interleukin (IL) 1β and transforming growth factor (TGF) β1, respectively. Mechanical stimulation was applied 4 hours a day for a total of 72 hours.

The performance measurements included the evaluation of

• ingredients related to the extracellular matrix (ECM),
• angiogenic factors,
• inflammatory and fibrogenic markers,

at the level of gene and protein expression.

Use of vibration in the study

Mechanical stimulation was carried out using a phonomimetic bioreactor developed by the authors of the presented article. Briefly, the flexible bottom 6-well hVFF plates were oscillated by the aerodynamic pressure fluctuations induced by the speaker placed beneath the plate. Immortalized hVFF was vibrated for three days as this time corresponds to the acute inflammatory phase. The pattern of stimulation, linear sinusoidal sound in the frequency range from 50 to 250 Hz, corresponded to the frequency range of the laryngeal sound in humans. The displacement of the membrane in the center of the well depended on the frequency used and averaged 82 ± 5 µm. The input voltage on the loudspeaker was set to 1.1 volts and served as a measure of the severity of the vibration. In order to exclude any interference, cells assigned to the control group – static, without vibration – were kept in a separate incubator.

Results

In inflammatory conditions, the pro-inflammatory cytokine IL11, as well as the myofibroblast marker alpha smooth muscle actin (α-SMA), promoting scarring, were significantly reduced after the application of vibration.

Moreover, the desired anti-fibrotic component of the ECM, hyaluronic acid, was increased after administration of cytokines and did not decrease after vibration.

Comment

Since human VF biopsies for molecular biology studies are unethical, the authors of this report have created, based on previous publications, a new in vitro model to study the effect of mechanical vibrations (simulating VF vibrations during speech) on inflammatory hVFF (simulating phonotrauma; activated fibroblasts can be responsible for fibrogenesis, i.e. fibrosis, scarring) and, by comparison, on normal hVFF.

The presented experiments revealed the effect of vibration stress on hVFF in inflammation. They show that elevated levels of some pro-inflammatory/ fibrogenic factors can be alleviated by vibration. Therefore, clinical trials that recommend early voice activation after an acute event have been confirmed. However, due to the obvious limitations of in vitro studies, further studies should be carried out to confirm the obtained observations, initially using still safe in vitro models, such as in vitro 3D modeling.

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