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Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold’s lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFβ-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device.

Objectives To explore the isolated or combined effects of cigarette smoke extract (CSE) and vibration on human vocal fold fibroblasts (hVFF) in an in vitro setting in order to elucidate their influence in the pathophysiology of Reinke's edema (RE). Study design Immortalized hVFF were exposed to CSE or control medium under static or vibrational conditions. A phonomimetic bioreactor was used to deliver vibrational patterns to hVFF over a period of 5 days. Methods Cytotoxicity was quantified using a lactate dehydrogenase assay. We employed reverse transcription–quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and Magnetic Luminex(R) assays (R&D Systems, Minneapolis, MN) to assess the influence on extracellular matrix production, fibrogenesis, inflammation, and angiogenesis. Results We observed significant changes induced by CSE alone (hyaluronic acid, matrix metalloproteinase 1, Interleukin‐8, cyclooxygenase [COX]1, COX2, vascular endothelial growth factor [VEGF]D), as well as settings in which only the combination of CSE and vibration led to significant changes (transforming growth factor beta 1, VEGFA, VEGFC). Also, CSE‐induced levels of COX2 were only significantly reduced when vibration was applied. Conclusion We were able to explore the cellular effects of CSE and vibration on hVFF by employing a phonomimetic bioreactor. Whereas cigarette smoke is generally accepted as a risk factor for RE, the role of vibration remained unclear as it is difficult to study in humans. Our data showed that some genes and proteins in the pathophysiological context of RE were only affected when CSE in combination with vibration was applied. Level of Evidence NA Laryngoscope, 131:E547–E554, 2021

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.

The voice disorder Reinke’s edema (RE) is a smoking- and voice-abuse associated benign lesion of the vocal folds, defined by an edema of the Reinke’s space, accompanied by pathological microvasculature changes and immune cell infiltration. Vocal fold fibroblasts (VFF) are the main cell type of the lamina propria and play a key role in the disease progression. Current therapy is restricted to symptomatic treatment. Hence, there is an urgent need for a better understanding of the molecular causes of the disease. In the present study, we investigated differential expression profiles of RE and control VFF by means of RNA sequencing. In addition, fast gene set enrichment analysis (FGSEA) was performed in order to obtain involved biological processes, mRNA and protein levels of targets of interest were further evaluated. We identified 74 differentially regulated genes in total, 19 of which were upregulated and 55 downregulated. Differential expression analysis and FGSEA revealed upregulated genes and pathways involved in extracellular matrix (ECM) remodeling, inflammation and fibrosis. Downregulated genes and pathways were involved in ECM degradation, cell cycle control and proliferation. The current study addressed for the first time a direct comparison of VFF from RE to control and evaluated immediate functional consequences.

The vast majority of voice disorders is associated with changes of the unique, but delicate, human vocal fold mucosa. The ability to develop new effective treatment methods is significantly limited by the physical inaccessibility and the extremely rare occasions under which healthy tissue biopsies can be obtained. Therefore, the interest in laryngological research has shifted to human oral (buccal) mucosa, a similar and more easily available tissue. The harvesting process is less invasive and accompanied with faster healing and less scarring, compared to vocal fold mucosa. Here we report a descriptive proteomic comparison of paired human buccal and vocal fold mucosa by high‐resolution mass spectrometry (CID‐MS/MS). Our study identified a total of 1575 proteins detected within both tissues that are highly consistent in several crucial biological processes, cellular components, and molecular functions. Hence, our proteomic analysis will provide a fundamental resource for the laryngological research community.

Zusammenfassung Hintergrund Trotz erheblicher Fortschritte in der laryngologischen Forschung gibt es eine Reihe von (benignen) Stimmlippenerkrankungen, die kausal nicht therapierbar sind. Das liegt an der eingeschränkten Zugänglichkeit sowie der sensiblen Mikroarchitektur der Stimmlippen, die nicht auf zellulärer Ebene erforscht werden können. Das pathophysiologische Verständnis endet dadurch häufig in der makroskopischen Ebene, die Folgen von Interventionen werden großteils endoskopisch oder mit indirekten Methoden evaluiert. Fragestellung Im nachfolgenden Beitrag stellen die Autor(inn)en biotechnologische State-of-the-Art-Methoden vor, die in der laryngologischen Forschung Anwendung finden, verbunden mit praktischen Beispielen. Ergebnisse Tierversuche und Zellkulturexperimente haben in den letzten Jahren zu einer signifikanten Wissenserweiterung beigetragen, dies insbesondere in den Bereichen Stimmlippeninflammation und -narbenbildung. Dem Stimmlippenfibroblasten, als wichtigstem zellulärem Bestandteil der Lamina propria, kommt dabei eine zentrale Rolle zu. Schlussfolgerungen Mittlerweile besteht bei einigen Krankheitsbildern ein tieferes Verständnis von Makroanatomie und Makropathophysiologie als je zuvor. In-vitro-Versuche zeigten beispielsweise, dass Stimmlippenfibroblasten in einem inflammatorischen Setting weniger profibrotische und proinflammatorische Zytokine sezernierten, wenn sie Vibrationen ausgesetzt sind. Umgesetzt auf die Klinik könnte das bedeuten, dass eine frühe Stimmaktivierung nach operativen Eingriffen an den Stimmlippen zu besserer Heilung und besseren stimmlichen Ergebnissen führt. Unsere Vision lautet, dass die molekulare Laryngologie ein gesichertes Fundament an Wissen bereitstellen soll, auf das in weiterer Folge klinische Studien aufgebaut werden können.

It is well known that the phonatory process changes during the life span. However, detailed investigations on potential factors concerned are rare. To deal with this issue, we performed extended biomechanical, macro anatomical, and histological analyses of the contributing laryngeal structures in ex vivo juvenile sheep models. Altogether twelve juvenile sheep larynges were analyzed within the phonatory experiments. Three different elongation levels and 16 different flow levels were applied to achieve a large variety of phonatory conditions. Vocal fold dynamics and acoustical and subglottal signals could be analyzed for 431 experimental runs. Subsequently, for six juvenile larynges microcomputed tomography following virtual 3D reconstruction was performed. The remaining six juvenile larynges as well as six ex vivo larynges from old sheep were histologically and immunohistologically analyzed. Results for juveniles showed more consistent dynamical behavior compared to old sheep larynges due to vocal fold tissue alterations during the life span. The phonatory process in juvenile sheep seems to be more effective going along with a greater dynamic range. These findings are supported by the histologically detected higher amounts of elastin and hyaluronic acid in the lamina propria of the juvenile sheep. The 3D reconstructions of the thyro-arytenoid muscles (TAM) showed a symmetrical shape. Intraindividual volume and surface differences of the TAM were small and comparable to those of aged sheep. However, TAM dimensions were statistically significant smaller for juvenile larynges. Finally, topographical landmarks were introduced for later comparison with other individuals and species. This work resulted in detailed functional, immunohistological, and anatomical information that was not yet reported. This data will also provide reference information for therapeutic strategies regarding aging effects, e.g. laryngeal muscle treatment by functional electrical stimulation.