Vocal Fold Fibroblasts Research Articles

HGF and bFGF Secreted by Adipose-Derived Mesenchymal Stem Cells Revert the Fibroblast Phenotype Caused by Vocal Fold Injury in a Rat Model

To investigate how adipose-derived mesenchymal stem cells (ADSCs), secreted hepatocyte growth factor (HGF), and basic fibroblast growth factor (bFGF) affect the fibroblast phenotype after vocal fold injury. We cultured primary normal (uninjured) and injured vocal fold fibroblasts (VFFs). A transwell co-culture system of ADSCs and injured VFFs was constructed in vitro, then the effects of HGF or bFGF were inhibited. The proliferation, extracellular matrix (ECM) secretion and transformation of VFFs were observed. Compared with uninjured VFFs, the secretion of collagen by injured VFFs increased significantly, hyaluronan synthase 1 (HAS1) secretion decreased, and VFF transformation increased significantly. After co-culture with ADSCs, the proliferation of VFFs was accelerated and the transformation was inhibited. Co-culture inhibited the expression of type I and III collagen and promoted the expression of HAS1. When HGF or bFGF secretion was inhibited, the proliferation of injured VFFs was inhibited. The inhibitory effect on collagen was reduced by both groups, but this was more obvious with the anti-HGF group. The anti-bFGF group had a more prominent effect on HAS1 secretion after injury than the anti-HGF group but the difference was not statistically significant. The inhibition of the transformation of injured VFFs was reduced while α-smooth muscle actin was upregulated, which was more obvious with the anti-HGF group. ADSCs and secreted HGF and bFGF can revert the fibroblast phenotype caused by vocal fold injury. The effects of HGF are more significant than bFGF on collagen secretion and the transformation of VFFs into myofibroblasts. However, bFGF is more effective than HGF in upregulating HAS1.

Effect of Urban Particulate Matter on Vocal Fold Fibrosis through the MAPK/NF-κB Signaling Pathway.

Particulate matter (PM) is an environmental exposure factor that adversely affects human health. PM is a risk factor for various diseases. However, the mechanism by which PM affects the vocal folds (VF) has not yet been evaluated. Thus, we investigated the cytotoxic effects of PM on human vocal fold fibroblasts (hVFF) and the underlying signaling pathways. hVFF were isolated from human VF. The effect of PM on hVFF, and the underlying mechanism, were analyzed using Western blot, quantitative real-time polymerase chain reaction, and flow cytometry. In addition, a histological evaluation was performed in animal experiments. Cell proliferation decreased after the PM treatment. PM increased the expression of interleukin (IL)-6 and IL-1β. The generation of reactive oxygen species (ROS) in PM-treated hVFF and subsequent activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways were confirmed. Furthermore, PM increased the expression of fibrosis-related markers and induced the accumulation of collagen in the extracellular matrix. As a result, PM exposure significantly enhances the inflammatory response on VF through the ROS-mediated activation of the MAPK and NF-κB signaling pathways. In addition, PM promotes differentiation into myofibroblasts and induces fibrosis. These results suggest that PM triggers an inflammatory reaction through ROS production and causes VF fibrosis.

Core-Shell Microfibers via Bioorthogonal Layer-by-Layer Assembly.

A new technique is described for the construction of core-shell microfibers for biomedical applications. Fibrous scaffolds were fabricated by electrospinning, followed by covalent layer-by-layer deposition based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctenes (TCOs). Electrospun poly(ε-caprolactone) (PCL) scaffolds were subjected to surface modifications to install tetrazine groups. The scaffolds were iteratively submerged in aqueous solutions of TCO-modified hyaluronic acid (HA-TCO) and tetrazine-modified hyaluronic acid (HA-Tz), resulting in the controlled growth of a cross-linked HA gel around individual microfibers. Integrin-binding motifs were covalently attached to the surface of the microfibers using TCO-conjugated RGD peptide. The scaffolds fostered the attachment and growth of primary porcine vocal fold fibroblasts without a significant induction of the myofibroblast phenotype. Stimulation with transforming growth factor beta (TGF-β) moderately enhanced fibroblast activation, and inhibition of the Rho/ROCK signaling pathway using Y27632 further decreased the expression of myofibroblastic markers. The bioorthogonally assembled scaffolds with a stiff PCL core and a soft HA shell may find application as therapeutic implants for the treatment of vocal fold scarring.

Exploring the Pathophysiology of Reinke's Edema: The Cellular Impact of Cigarette Smoke and Vibration

ObjectivesTo 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 designImmortalized 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.MethodsCytotoxicity 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.ResultsWe 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.ConclusionWe 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 EvidenceNA Laryngoscope, 131:E547–E554, 2021

The Antiproliferative and Antifibrotic Effects of Cisplatin on Primary Human Vocal Fold Fibroblasts

Objective: Vocal fold scarring and laryngeal stenosis are major clinical challenges. Current drugs do not efficiently reduce scarring. We examined the antiproliferative and antifibrotic effects of cisplatin on primary human vocal fold fibroblasts (HVFFs). Methods: HVFFs were cultured in vitro and identified by immunocytochemistry. The relative viability of HVFFs was analyzed by Cell Counting Kit-8 assays (CCK-8). The fibrogenic phenotype was induced by transforming growth factor-β₁ (TGF-β₁) and reversed by cisplatin as shown by immunocytochemistry. Real-time PCR and Western blotting assessed collagen III and I. Western blotting for Smad2, p-Smad2, Smad-3, p-Smad3 and caspase-3 were performed. Results: CCK-8 results showed that cisplatin decreased the relative viability of HVFFs, and Western blots revealed elevation of the apoptosis-related protein caspase-3 in HVFFs. Cisplatin treatment reduced α-smooth muscle actin staining intensity in the presence of TGF-β₁. Real-time PCR revealed the downregulation of collagen III and I in cisplatin-treated HVFFs. The TGF-β₁-induced increased fibrogenic protein levels were decreased by cisplatin. Reduced levels were detected at late time points. Conclusions: Cisplatin induces antiproliferative and antifibrotic alterations in HVFFs. Cisplatin may prevent postoperative vocal fold scarring and laryngeal stenosis in patients treated with CO₂ laser microsurgery and undergoing delayed wound healing.

Antioxidant Properties of Tonsil-Derived Mesenchymal Stem Cells on Human Vocal Fold Fibroblast Exposed to Oxidative Stress

The therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) has been proved in several in vitro and in vivo models based on their antioxidative capacity. Oxidative stress is involved in the formation of vocal fold scars and the aging of vocal folds. However, few studies have examined the direct correlation between oxidative damage and reconstitution of extracellular matrix (ECM) in the vocal fold fibrosis. We, therefore, sought to investigate the impact of oxidative stress on cell survival and ECM production of human vocal fibroblasts (hVFFs) and the protective effects elicited by TMSCs against oxidative damages in hVFFs. hVFFs were exposed to different concentrations of tert-butyl hydroperoxide in the presence or absence of TMSCs. Cell viability and reactive oxygen species (ROS) production were assessed to examine the progression of oxidative stress in vitro. In addition, expression patterns of ECM-associated factors including various collagens were examined by real-time PCR and immunocytochemical analysis. We found that both cell viability and proliferation capacity of hVFFs were decreased following the exposure to tBHP in a dose-dependent manner. Furthermore, tBHP treatment induced the generation of ROS and reactive aldehydes, while it decreased endogenous activity of antioxidant enzymes in hVFF. Importantly, TMSCs could rescue these oxidative stress-associated damages of hVFFs. TMSCs also downregulated tBHP-mediated production of proinflammatory cytokines in hVFFs. In addition, coculture with TMSC could restore the endogenous matrix metalloproteinase (MMP) activity of hVFFs upon tBHP treatment and, in turn, reduce the oxidative stress-induced ECM accumulation in hVFFs. We have, therefore, shown that the changes in hVFF proliferative capacity and ECM gene expression induced by oxidative stress are consistent with in vivo phenotypes observed in aging vocal folds and vocal fold scarring and that TMSCs may function to reduce oxidative stress in aging vocal folds.

The Effects of Photobiomodulation on Vocal Fold Wound Healing: In Vivo and In Vitro Studies.

Background: Photobiomodulation (PBM) is increasingly used in dermatology and dentistry due to its benefit of promoting wound healing and relieving pain; however, there is no corresponding research report on the application of PBM to vocal fold wound healing. Objective: To assess the potential wound-healing effects of PBM on the vocal folds via in vivo and in vitro experiments. Materials and methods: In in vitro study, vocal fold fibroblasts (VFFs) were irradiated under a diode laser with wavelength of 635 nm at energy density of 8 J/cm2. The Cell Counting Kit-8 (CCK-8) assay was used to study the viability of VFFs, and the gene expressions of COL1A2, COL3A1, IL-6, HAS2, and COX-2 were investigated by real-time polymerase chain reaction (RT-PCR). In in vivo study, 15 rabbits were used. Lamina propria of the left vocal folds of 12 rabbits was unilaterally stripped, and 6 of them were treated with PBM. The remaining three rabbits served as normal controls. After 3 months, all animals were sacrificed to obtain histological results. We used laryngoscope to record images of the healing phase. Results: Irradiation with energy density of 8 J/cm2 resulted in a 2.8% increase in cell proliferation (p < 0.05). However, the difference between the experimental and the control group became larger after 48 and 72 h of subsequent irradiation. RT-PCR results showed that the expression of COL1A2, COL3A1, and HAS2 was higher, and the expression of IL-6 and COX-2 was lower. Histological examination showed that, compared with the injury group, hyaluronic acid (HA) increased significantly, collagen deposition decreased, and the configuration of collagen was more organized after PBM treatment. Conclusions: PBM can inhibit inflammatory reaction and promote the secretion of HA to decrease the deposition of collagen and regenerate vocal fold tissue without scar.

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema*[S

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.

Carbon nanotube composite hydrogels for vocal fold tissue engineering: Biocompatibility, rheology, and porosity

Porous composite hydrogels were prepared using glycol chitosan as the matrix, glyoxal as the chemical crosslinker, and carbon nanotubes (CNTs) as the fibers. Both carboxylic and hydroxylic functionalized CNTs were used. The homogeneity of CNTs dispersion was evaluated using scanning electron microscopy. Human vocal fold fibroblasts were cultured and encapsulated in the composite hydrogels with different CNT concentrations to quantify cell viability. Rheological tests were performed to determine the gelation time and the storage modulus as a function of CNT concentration. The gelation time tended to decrease for low concentrations and increase at higher concentrations, reaching a local minimum value. The storage modulus obeyed different trends depending on the functional group. The porosity of the hydrogels was found to increase by 120% when higher concentrations of carboxylic CNTs were used. A high porosity may promote cell adhesion, migration, and recruitment from the surrounding native tissue, which will be investigated in a future work aiming at applying this injectable biomaterial for vocal fold tissue regeneration.

Liquid-type non-thermal atmospheric plasma ameliorates vocal fold scarring by modulating vocal fold fibroblast.

Injection laryngoplasty is a widely used therapeutic option for drug delivery into vocal folds (VFs). Efficient injectable materials are urgently needed for treating intractable VF disease. Liquid-type non-thermal atmospheric plasma (LTP) has been found to be useful for various biological applications, including in regenerative medicine. We evaluated the effects of LTP on VF regeneration. Migration and matrix metalloproteinase-2 expression of lipopolysaccharide (LPS)-treated human vocal fold-derived mesenchymal stem cells (VF-MSCs) were enhanced by LTP treatment. LTP treatment not only ameliorated nuclear factor-κB and interleukin-6 activation, induced by LPS treatment, but also the increased manifestation of α-smooth muscle actin and fibronectin, induced by transforming growth factor-ß. In a rabbit VF scarring animal model, histological analyses showed increased hyaluronic acid deposition and decreased collagen accumulation after LTP injection. Videokymographic analysis showed more improved vibrations in LTP-treated VF mucosa compared to those in non-treated group. In conclusion, LTP treatment enhanced the recruitment and activation of VF-MSCs. Regulated extracellular matrix (ECM) synthesis and eventual functional improvement of scarred VFs were observed upon LTP treatment. The results of this study suggest that LTP injection can enhance wound healing and improve functional remodeling following VF injury.Impact statementVoice disorder has a significant impact on life quality, and one of the major causes of this voice disorder is vocal fold scarring. Therefore, various approaches have been tried to treat for voice disorder. However, no method has satisfied all requirements until now. Plasma medicine, which involves the medical application of plasma, is a rapidly developing field. We have confirmed that liquid-type plasma improved vocal fold scarring by mobilizing and activating vocal fold fibroblast. In conclusion, liquid-type plasma is a potential therapeutic agent for promoting vocal fold scarring through simple injection and it may be an alternative therapeutic agent for the current situation to treat voice disorder.

Magnetic Tissue Engineering of the Vocal Fold Using Superparamagnetic Iron Oxide Nanoparticles.

Losing one's ability to speak, because of tissue deficiency at the vocal fold (VF), leads to serious impairment in the quality of life. Until now, there is no successful approach for regenerating the VF. The aim of this study was to show the advantage of magnetic nanoparticles in the generation of scaffold-free three-dimensional (3D) VF cell constructs by magnetic tissue engineering (MTE). Rabbit VF fibroblasts were used to establish MTE: after cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs), cells can be controlled with a magnetic field thereby forming solid 3D cell structures. To transfer this method into human cells, SPIONs were adapted accordingly and tested for their influence on human VF (hVF) cells and for their ability to perform MTE with hVF cells. Of interest, the cell number and the magnet's shape influence the form of the rabbit VF cell construct. After successful characterization of hVF cells, biocompatibility analyses revealed no significant influence of SPIONs on them, thus 3D hVF cell constructs could be successfully generated by MTE. These basic results are important to develop MTE as an innovative method to regenerate functional VFs. We expect that in vivo studies, including MTE as an elegant, far-field controlled and touchless technology, will translate MTE VF bioconstructs into reconstructive laryngeal medicine. Impact Statement This study aims at nanotechnology for regenerative medicine by magnetic tissue engineering (MTE). New approaches for vocal fold (VF) reconstruction are desperately needed. Superparamagnetic iron oxide nanoparticles offer innovative, scaffold-free potentials for tissue engineering: MTE. By using MTE we could generate functional multilayered human VF cell constructs, which can consequently be used to regenerate the voice in patients with VF injuries.

Tissue specific human fibroblast differential expression based on RNAsequencing analysis

BackgroundPhysical forces, such as mechanical stress, are essential for tissue homeostasis and influence gene expression of cells. In particular, the fibroblast has demonstrated sensitivity to extracellular matrices with assumed adaptation upon various mechanical loads. The purpose of this study was to compare the vocal fold fibroblast genotype, known for its unique mechanically stressful tissue environment, with cellular counterparts at various other anatomic locales to identify differences in functional gene expression profiles.ResultsBy using RNA-seq technology, we identified differentially expressed gene programs (DEseq2) among seven normal human fibroblast primary cell lines from healthy cadavers, which included: vocal fold, trachea, lung, abdomen, scalp, upper gingiva, and soft palate. Unsupervised gene expression analysis yielded 6216 genes differentially expressed across all anatomic sites. Hierarchical cluster analysis revealed grouping based on anatomic site origin rather than donor, suggesting global fibroblast phenotype heterogeneity. Sex and age-related effects were negligible. Functional enrichment analyses based on separate post-hoc 2-group comparisons revealed several functional themes within the vocal fold fibroblast related to transcription factors for signaling pathways regulating pluripotency of stem cells and extracellular matrix components such as cell signaling, migration, proliferation, and differentiation potential.ConclusionsHuman fibroblasts display a phenomenon of global topographic differentiation, which is maintained in isolation via in vitro assays. Epigenetic mechanical influences on vocal fold tissue may play a role in uniquely modelling and maintaining the local environmental cellular niche during homeostasis with vocal fold fibroblasts distinctly specialized related to their anatomic positional and developmental origins established during embryogenesis.

Reversal of Vocal Fold Mucosal Fibrosis Using siRNA against the Collagen-Specific Chaperone Serpinh1

Vocal fold (VF) mucosal fibrosis results in substantial voice impairment and is recalcitrant to current treatments. To reverse this chronic disorder, anti-fibrotic therapies should target the molecular pathology of aberrant collagen accumulation in the extracellular matrix. We investigated the therapeutic potential of siRNA against Serpinh1, a collagen-specific chaperone that enables cotranslational folding and assembly of procollagens in the endoplasmic reticulum. We implemented a previously validated siRNA construct, conducted transfection experiments using in vitro and in vivo rat models, and measured knockdown efficiency, dose responses, delivery strategies, and therapeutic outcomes. Liposome-mediated delivery of Serpinh1-siRNA downregulated collagen production in naive and scar VF fibroblasts as well as naive VF mucosa; moreover, sustained Serpinh1 knockdown in fibrotic VF mucosa reversed scar-associated collagen accumulation within 4 weeks. Analysis of therapeutic effects at the transcriptome level showed evidence of cell cycle upregulation, catabolism, matrix disassembly, and morphogenesis. These findings indicate that Serpinh1-siRNA holds potential as a molecular therapy for chronic VF mucosal fibrosis.

Investigation of surgical adhesives for vocal fold wound closure.

Surgical adhesives are increasingly used for vocal fold microsurgery to assist wound closure and reduce the risks of scar formation. Currently used vocal fold adhesives such as fibrin glue, however, have thus far not been found to promote wound closure or reduce scarring. The objectives of this study were to investigate the mechanical strength and the cytotoxicity of three commercially available adhesives (Glubran 2, GEM, Viareggio, Italy; BioGlue, CryoLife, Kennesaw, GA; and Tisseel, Baxter Healthcare, Deerfield, IL) for vocal fold wound closure. Shear and tension tests were performed on 150 porcine larynges. The cytotoxicity of the adhesives to immortalized human vocal fold fibroblasts was investigated using neutral red uptake assays. The average shear adhesive strength for Tisseel, BioGlue, and Glubran 2 was 13.86 ± 5.03 kilopascal (kPa), 40.92 ± 17.94 kPa, and 68.79 ± 13.29 kPa, respectively. The tensile adhesive strength for Tisseel, BioGlue, and Glubran 2 was 10.70 ± 6.42 kPa, 34.27 ± 12.59 kPa, and 46.67 ± 12.13 kPa, respectively. The vocal fold cell viabilities in extracts of Tisseel, BioGlue, and Glubran 2 were 99.27%, 43.05%, and 1.79%, respectively. There was a clear tradeoff between adhesive strength and toxicity. The maximum failure strength in shear or tension of the three surgical adhesives ranked from strongest to the weakest was: 1) Glubran 2, 2) BioGlue, and 3) Tisseel. Tisseel was found to be the least toxic of the three adhesives, whereas Glubran 2 was the most toxic. NA Laryngoscope, 129:2139-2146, 2019.

Development and validation of a novel phonomimetic bioreactor.

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.

The role of pepsin in epithelia-mesenchymal transition in idiopathic subglottic stenosis.

Idiopathic subglottic stenosis (iSGS) is commonly characterized by laryngeal fibrosis thought to arise by epithelia-mesenchymal transition (EMT) induced by chronic inflammation. Pepsin is a potent inducer of inflammation in the airways during chronic laryngopharyngeal reflux and has been observed in the subglottic mucosa of patients with iSGS, absent in normal mucosa. The aim of this study was to examine the effect of pepsin on mechanisms of EMT in laryngeal cells with implications for iSGS. In vitro translational research study. Human laryngeal epithelial cell cultures were exposed to 0.1 mg/mL or 1.0 mg/mL pepsin at pH7 for 24 and 48 hours, or media pH5 ± 0.1 mg/mL pepsin for 10 minutes and harvested after 24 and 48 hours. EMT marker expression was measured by qPCR and enzyme-linked immunosorbent assays. Wound-healing scratch assay was performed on immortalized human vocal fold fibroblasts pretreated with media pH5 ± 0.1 mg/mL pepsin (10 minutes) or continuously treated with media pH7 ± 0.1 to 1 mg/mL pepsin for 24 hours. Pepsin yielded no effect on MMP1, MMP9, FN1, COL1A1, HAS2, or CDH1 gene expression or matrix metalloproteinase-9 or fibronectin protein expression, either alone or in the presence of weak acid. Pepsin and/or acid produced no effect on fibroblast migration. Whereas pepsin has been shown to be present in the subglottic mucosa of patients with iSGS, this in vitro acute exposure investigation does not provide evidence of a direct causal role for development of fibrosis in subglottic epithelial cell cultures. NA. Laryngoscope, 130:154-158, 2020.

Incorporation of types I and III collagen in tunable hyaluronan hydrogels for vocal fold tissue engineering

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. Statement of SignificanceVoice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.

Dual growth factor-immobilized bioactive injection material for enhanced treatment of glottal insufficiency

With increasing demand for treatment of glottal insufficiency, several injection materials have been examined. However, biological resorption, degradation of injected materials, and the subsequent need to perform multiple injections still remain major clinical problems. In this study, we fabricated two different growth factor (GF) [single basic fibroblast growth factor (bFGF), single hepatocyte growth factor (HGF), or dual bFGF/HGF]-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres. These materials were investigated for their potential use as bioactive injection laryngoplasty agents. HGF was found to be continuously released over 20 days and the bFGF was found to be continuously released over 25 days, as demonstrated by ELISA assay. Human vocal fold fibroblasts (hVFFs) showed significantly higher proliferative ability on dual GF-immobilized microspheres. GF-immobilized microspheres (bFGF, HGF, and dual GF) were injected into paralyzed vocal folds of New Zealand white rabbits. Through endoscopic observation and H&E staining, we identified that the microspheres remained localized at the injection site, resulting in constant volume augmentation of the paralyzed vocal fold without significant loss of the initial volume after 4 weeks. The expression of genes related to the extracellular matrix (ECM) in the vocal fold was upregulated by dual GF-immobilized microspheres. Furthermore, dual GF-immobilized microspheres inhibited muscle degeneration and upregulation of myogenic-related genes. In conclusion, dual GF-immobilized microspheres passively augmented the volume of the paralyzed vocal fold while actively inducing ECM synthesis at the injected vocal fold and preserving muscle tissue. Dual GF-immobilized microspheres could be a new and promising injection material for paralyzed vocal folds. Statement of SignificanceLimitation of prolonged augmentation of vocal fold and degeneration of vocal fold tissue still remain as major clinical problems in the treatment of vocal fold paralysis. Herein, we fabricated the polycaprolactone (PCL)/Pluronic F127 microspheres to augment volume of paralyzed vocal folds. On top of that, we additionally immobilized the growth factors (bFGF, HGF, or dual bFGF/HGF) on the surface of these microspheres. We highlight the efficacy of the dual GF-immobilized microspheres which augmented the volume of the paralyzed vocal fold passively, induced ECM synthesis actively at the injected vocal fold and preserved laryngeal muscle tissue. Our results suggest that the dual GF-immobilized microsphere could be a new promising injection material for injection laryngoplasty to treat paralyzed vocal fold.

Mechanotransduction of vocal fold fibroblasts and mesenchymal stromal cells in the context of the vocal fold mechanome

The design of cell-based therapies for vocal fold tissue engineering requires an understanding of how cells adapt to the dynamic mechanical forces found in the larynx. Our objective was to compare mechanotransductive processes in therapeutic cell candidates (mesenchymal stromal cells from adipose tissue and bone marrow, AT-MSC and BM-MSC) to native cells (vocal fold fibroblasts-VFF) in the context of vibratory strain. A bioreactor was used to expose VFF, AT-MSC, and BM-MSC to axial tensile strain and vibration at human physiological levels. Microarray, an empirical Bayes statistical approach, and geneset enrichment analysis were used to identify significant mechanotransductive pathways associated with the three cell types and three mechanical conditions. Two databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes) were used for enrichment analyses. VFF shared more mechanotransductive pathways with BM-MSC than with AT-MSC. Gene expression that appeared to distinguish the vibratory strain condition from polystyrene condition for these two cells types related to integrin activation, focal adhesions, and lamellipodia activity, suggesting that vibratory strain may be associated with cytoarchitectural rearrangement, cell reorientation, and extracellular matrix remodeling. In response to vibration and tensile stress, BM-MSC better mimicked VFF mechanotransduction than AT-MSC, providing support for the consideration of BM-MSC as a cell therapy for vocal fold tissue engineering. Future research is needed to better understand the sorts of physical adaptations that are afforded to vocal fold tissue as a result of focal adhesions, integrins, and lamellipodia, and how these adaptations could be exploited for tissue engineering.

Characterization of aged rat vocal fold fibroblasts.

To elucidate the aging physiology of the vocal folds, we examined the characters of aged vocal fold fibroblasts (VFFs) in various conditions. In vitro study. VFFs from young (12-week-old) and aged (19-month-old) Sprague-Dawley rats were compared. Proliferative capacity, ratio of myofibroblast to fibroblast, myofibroblast function, and extracellular matrix production were examined in the following conditions: naïve, basic fibroblast growth factor (bFGF) supplemented, and hepatocyte growth factor (HGF) supplemented. Aged VFFs demonstrated reduced proliferation by cell counting, though the ratio of Ki-67-positive cells showed no difference. Aged VFFs exhibited an increased expression of α-smooth muscle actin (α-SMA); however, they demonstrated no enhanced contractile ability in a gel contraction assay. Type I collagen protein was increased age dependently, accompanied with decreased Mmp1 and unchanged Col1a1 transcription. Type I collagen protein and α-SMA represented quite similar reduction patterns to bFGF or HGF administration. The following possible characteristics of aged VFFs were implied: long duration of mitosis, increased myofibroblast population size with certain dysfunctions, reduced type I collagen turnover, and correlation between α-SMA expression and type I collagen metabolism. Further investigations of these features will help to clarify presbyphonia's pathology and establish treatment strategies. NA Laryngoscope, 129:E94-E101, 2019.