Towards an in vitro fibrogenesis model of human vocal fold scarring

Abstract

BackgroundVocal fold (VF) scarring remains a therapeutic dilemma and challenge in modern laryngology. To facilitate corresponding research, we aimed to establish an in vitro fibrogenesis model employing human VF fibroblasts (hVFF) and the principles of macromolecular crowding (MMC).MethodsFibrogenesis was promoted by addition of transforming growth factor-β1 to standard medium and medium containing inert macromolecules (MMC). Hepatocyte growth factor (HGF) and Botox type A were tested for their antifibrotic properties in various doses. Experiments were analyzed with respect to the biosynthesis of collagen, fibronectin, and α-smooth muscle actin using immunofluorescence, silver stain and western blot.ResultsMMC led to favourable enhanced deposition of collagen and other extracellular matrix components, reflecting fibrotic conditions. Low doses of HGF were able to dampen profibrotic effects. This could not be observed for higher HGF concentrations. Botox type A did not show any effects.ConclusionBased on the principles of MMC we could successfully establish a laryngeal fibrogenesis model employing hVFF. Our finding of dose-dependent HGF effects is important before going into clinical trials in humans and has never been shown before. Our model provides a novel option to screen various potential antifibrotic compounds under standardized conditions in a short time.Electronic supplementary materialThe online version of this article (10.1007/s00405-018-4922-7) contains supplementary material, which is available to authorized users.

Pathophysiology of Fibrosis in the Vocal Fold: Current Research, Future Treatment Strategies, and Obstacles to Restoring Vocal Fold Pliability

“…Branco et al evaluated the myofibroblast profile isolated from normal and scarred human VF and suggested that VF fibroblast treated with TGFβ1 (myofibroblasts) appear to have similar phenotypic characteristics but different genotypic behavior compared to VF fibroblasts isolated from human VF scars [61]. Graupp et al established a laryngeal fibrogenesis model employing human VF fibroblasts based on the principle of the macromolecular crowding [62]. These excellent recent in vitro VF fibroblast models would accelerate the development of the novel treatment strategy for VF scars.…”

Macromolecular Crowding as a Tool to Screen Anti-fibrotic Drugs: The Scar-in-a-Jar System Revisited

“…Since the hallmark of fibrosis is the deposition of excessive amounts of collagen by activated fibroblasts (i.e., myofibroblasts) ( 29 , 30 ), MMC might be a helpful tool for cellular drug screening, as it markedly shortens the culture time at which collagen deposition becomes visible. Indeed, MMC has been used for this purpose ( 5 , 6 , 31 , 32 ), which is also known as the Scar-in-a-Jar system.…”

“…They are Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 10, dextran sulfate 500 (DxS500), carrageenan (CR), and polysodium-4-styrene sulfonate. Human fibroblasts used are WI-38 cells (embryonic lung fibroblasts) ( 3 , 4 , 6 , 31 , 38 ), WS-1 cells (embryonic dermal fibroblasts) ( 38 , 39 ), adult dermal fibroblasts ( 37 , 40 ), adult corneal fibroblasts (keratocytes) ( 34 – 36 ) and immortalized adult vocal fold fibroblasts ( 32 ). However, it should be stressed that it is the myofibroblast that is mainly responsible for the collagen deposition seen in fibrosis, not the fibroblast.…”

“…The latter can be achieved by adding the strong profibrotic cytokine TGFβ1 into the culture medium ( 41 , 42 ). The only reports that took this into consideration are those of Chen et al ( 6 , 31 ) and Graupp et al ( 32 ), with DxS500 and Fc70/400 as MMCs. However, Chen et al ( 6 , 31 ) used embryonic lung fibroblasts; it is known that embryonic fibroblasts are phenotypically different from adult fibroblasts, as scarring does not occur in the embryo ( 43 , 44 ).…”

Paracrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An In Vitro Fibrogenesis Model Of Human Vocal Fold Scarring

“…Other more experimental treatments such as administration of basic Fibroblast Growth Factor (bFGF) [9,10] or Hepatocyte Growth Factor (HGF) [11][12][13][14] have shown encouraging effects in vitro or in vivo on animals. However, only one phase I/II clinical trial assessed the safety and effectiveness of intracordal injection of a recombinant human HGF in patients with vocal fold scar or sulcus [15].…”