An in vitro scaffold‐free epithelial–fibroblast coculture model for the larynx

Objectives/hypothesisPhysiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1.Study designIn vitro.MethodsCell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor β1 (TGFβ1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin.ResultsFibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFβ1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture.ConclusionsThese data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.Level of evidenceNA Laryngoscope, 127:E185-E192, 2017

A Review of Hyaluronic Acid and Hyaluronic Acid-based Hydrogels for Vocal Fold Tissue Engineering

Vocal fold scarring is a common cause of dysphonia. Current treatments involving vocal fold augmentation do not yield satisfactory outcomes in the long term. Tissue engineering and regenerative medicine offer an attractive treatment option for vocal fold scarring, with the aim to restore the native extracellular matrix microenvironment and biomechanical properties of the vocal folds by inhibiting progression of scarring and thus leading to restoration of normal vocal function. Hyaluronic acid is a bioactive glycosaminoglycan responsible for maintaining optimum viscoelastic properties of the vocal folds and hence is widely targeted in tissue engineering applications. This review covers advances in hyaluronic acid-based vocal fold tissue engineering and regeneration strategies

Acute Acrolein Exposure Induces Impairment of Vocal Fold Epithelial Barrier Function.

Acrolein is a ubiquitous pollutant abundant in cigarette smoke, mobile exhaust, and industrial waste. There is limited literature on the effects of acrolein on vocal fold tissue, although there are clinical reports of voice changes after pollutant exposures. Vocal folds are responsible for voice production. The overall objective of this study was to investigate the effects of acrolein exposure on viable, excised vocal fold epithelial tissue and to characterize the mechanism underlying acrolein toxicity. Vocal fold epithelia were studied because they form the outermost layer of the vocal folds and are a primary recipient of inhaled pollutants. Porcine vocal fold epithelia were exposed to 0, 50, 100, 500, 900 or 1300 μM of acrolein for 3 hours; the metabolic activity, epithelial resistance, epithelial permeability, tight junction protein (occludin and claudin 3) expression, cell membrane integrity and lipid peroxidation were investigated. The data demonstrated that acrolein exposure at 500 μM significantly reduced vocal fold epithelial metabolic activity by 27.2% (p≤0.001). Incubation with 100 μM acrolein caused a marked increase in epithelial permeability by 130.5% (p<0.05) and a reduction in transepithelial electrical resistance (TEER) by 180.0% (p<0.001). While the expression of tight junctional protein did not change in acrolein-treated samples, the cell membrane integrity was significantly damaged with a 45.6% increase of lipid peroxidation as compared to controls (p<0.05). Taken together, these data provide evidence that acute acrolein exposure impairs vocal fold epithelial barrier integrity. Lipid peroxidation-induced cell membrane damage may play an important role in reducing the barrier function of the epithelium

The effects of vocal exertion on lung volume measurements and acoustics in speakers reporting high and low vocal fatigue.

PurposeVocal exertion is common and often results in reduced respiratory and laryngeal efficiency. It is unknown, however, whether the respiratory kinematic and acoustic adjustments employed during vocal exertion differ between speakers reporting vocal fatigue and those who do not. This study compared respiratory kinematics and acoustic measures in individuals reporting low and high levels of vocal fatigue during a vocal exertion task.MethodsIndividuals reporting low (N = 20) and high (N = 10) vocal fatigue participated in a repeated measures design study over 2 days. On each day, participants completed a 10-minute vocal exertion task consisting of repeated, loud vowel productions at elevated F0 sustained for maximum phonation time. Respiratory kinematic and acoustic measures were analyzed on the 1st vowel production (T0), and the vowels produced 2 minutes (T2), 5 minutes (T5), 7 minutes (T7), and 10 minutes (T10) into the vocal exertion task. Vowel durations were also measured at each time point.ResultsNo differences in respiratory kinematics were observed between low and high vocal fatigue groups at T0. As the vocal exertion task progressed (T2-T10), individuals reporting high vocal fatigue initiated phonation at lower lung volumes while individuals with low vocal fatigue initiated phonation at higher lung volumes. As the exertion task progressed, total lung volume excursion decreased in both groups. Differences in acoustic measures were observed, as individuals reporting high vocal fatigue produced softer, shorter vowels from T0 through T10.ConclusionsIndividuals reporting high vocal fatigue employed less efficient respiratory strategies during periods of increased vocal demand when compared with individuals reporting low vocal fatigue. Individuals reporting high vocal fatigue had shorter maximum phonation time on loud vowels. Further study should examine the potential screening value of loud maximum phonation time, as well as the clinical implications of the observed respiratory patterns for managing vocal fatigue

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 SIGNIFICANCE: Voice 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

Western blot analysis of occludin protein with or without acrolein exposure.

<p>Data represent Mean ± SE, n = 6, p = 0.337 as compared to controls. “C” represents control group and “A” represents acrolein treated group.</p

Cell membrane integrity assessed by LDH leakage.

<p>Data represents LDH release in extracellular medium normalized by the average in the control group, #: p<0.01 as compared to controls by paired t-test.</p

Expression levels of mRNAs encoding typical tight junctional proteins.

<p>Tissues were incubated with acrolein as the concentration indicated for 3 hours. The mRNA levels of occludin (A) and claudin3 (B) were determined by qPCR. Data represent Mean ± SE, n = 6 (p = 0.259 for occludin and p = 0.556 for claudin3) as compared to controls.</p

The metabolic activities of vocal fold tissue following acrolein exposure ex vivo.

<p>Epithelial metabolic activity was determined by the MTT assay. Tissues were incubated with acrolein as the concentration indicated for 3 hours. Data represent Mean ± SE, n = 7, *: p < 0.05, §: p < 0.001 as compared to controls.</p