Methodology for the Establishment of Primary Porcine Vocal Fold Epithelial Cell Cultures

Objective: A current lack of methods for epithelial cell culture significantly hinders our understanding of the role of the epithelial and mucus barriers in vocal fold health and disease. Our first objective was to establish reproducible techniques for the isolation and culture of primary porcine vocal fold epithelial cells. Our second objective was to evaluate the functional significance of cell cultures using an in vitro exposure to an inflammatory cytokine. Methods: Epithelial cells were isolated from porcine vocal folds and expanded in culture. Characterization of cultures was completed by immunostaining with markers for pan-cytokeratin (epithelial cells), vimentin (stromal cells), von Willebrand factor (endothelial cell), and MUC1 and MUC4 (mucin) glycoproteins. Established epithelial cell cultures were then exposed to the inflammatory cytokine tumor necrosis factor alpha (TNF-α) for 24-hours, and transcript expression of MUC1 and MUC4 was evaluated. Results: Reproducible, porcine vocal fold epithelial cell cultures, demonstrating cobblestone appearance characteristic of the typical morphology of epithelial cell cultures were created. Cells showed positive staining for pan-cytokeratin with limited expression of vimentin and von Willebrand factor. Epithelial cells also expressed MUC1 and MUC4. TNF-α significantly increased transcript expression of MUC4. Conclusion: Here, we present the first report of successful culture of primary porcine vocal fold epithelial cells. Cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus as well as the effects of common challenges, including inflammatory cytokines, on these barriers

Structural and Functional Vocal Fold Epithelial Integrity Following Injury

Objectives/Hypothesis: An intact epithelium is an important part of vocal fold defense. Damage to the epithelium can compromise vocal fold homeostasis and protection of the host tissue from viral and bacterial invasion. Elucidating the effects of damage on epithelial architectural and barrier integrity provides insight into the role of epithelium in protecting vocal folds. Using an animal model, we evaluated the time course of structural and functional epithelial restoration following injury. Study Design: Prospective, controlled animal study. Methods: Forty rats underwent surgery to remove vocal fold mucosa unilaterally. Larynges were harvested at five time intervals between 3 to 90 days postinjury and were prepared for histological and permeability analyses. Results: Rapid restoration of structural integrity was demonstrated by return of a multilayerd epithelium, intercellular junctions, and basement membrane at 5 days postinjury. Atypical epithelial permeability was observed up to 5 weeks postinjury. Conclusion: Restoration of epithelial barrier integrity lags epithelial structural restoration. Consequently, epithelial regeneration cannot be equated with return of functional barrier integrity. Rather, ongoing leakiness of regenerated epithelium indicates that vocal folds remain at risk for damage, pathogen invasion, and remodeling postinjury

Epidermal Growth Factor Mediated Healing in Stem Cell-derived Vocal Fold Mucosa

Background: The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. Materials and methods: A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation after injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, gefitinib. Results: Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 μm/h, ±2.63) compared with absence of exogenous EGF (7.1 μm/h ±2.84), but inhibited with concurrent addition of Gefitinib (5.2 μm/h, ±2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. Although not statistically significant, increased density of Ki67 staining in the epithelium adjacent to the scratch wound was observed after treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. Conclusions: Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair after injury

Voice Disorders

Goal Writing for the Speech-Language Pathologist and Special Educator is a practical guide on how to bridge the gap between the ability to assess, interpret, and analyze assessment information in order to derive functional goals for intervention. Contributed by experts in the field, this resource is appropriate for both novice and experienced clinicians in the field of Speech-Language Pathology and Special Education, as well as students learning to develop and write functional goals for different disorders and populations. Case scenarios available with each chapter provide real-world context for how to develop goals from the assessment process

Derivation of Epithelial Cells from Human Embryonic Stem Cells as an In Vitro Model of Vocal Mucosa

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here, we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air–liquid interface for 19–21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology, gene expression, and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and, therefore, offers a promising approach to studying vocal fold biology and biomechanics in health and disease

Perceptual ratings of vocal characteristics and voicing features in untreated patients with unilateral vocal fold paralysis

This study used visual analog scales to obtain perceptual ratings of features of voice production in subjects with unilateral vocal fold paralysis (UVFP), including clarity of laryngeal articulation, consistency of loudness across the utterance and the voiced/voiceless distinction. Recordings of repeated /i/, /isi/, and /izi/ from subjects diagnosed with UVFP and control subjects were randomly re-recorded, and then rated by five listeners. Significant differences in ratings (Smirnov test, p \u3c 0.01) were found between groups for aphonia , severity , clarity of articulation , overall loudness , consistency of loudness and amount of effort . Four of five raters agreed on the accuracy of /s/ or /z/ productions for only 54% of the samples from the subjects with UVFP. Voiceless and voiced cognates were equally likely to be rated as inaccurate. Results suggested that these variables were sensitive to changes in voice production resulting from paralysis, and may be useful in measuring treatment outcomes and spontaneous recovery of function. Learning outcomes: As a result of reading this manuscript the reader will (1) gain an understanding of types of perceptual scales and how to develop the set of vocal characteristics to be used in distinguishing patients with UVFP and those without, (2) learn which vocal characteristics listeners are able to use to successfully distinguish between patients with UVFP and those without and (3) understand the possible role for perceptual ratings in tracking changes in vocal characteristics in subjects over time following treatment or spontaneous recovery of function. © 2004 Elsevier Inc. All rights reserved

Classification for Animal Vocal Fold Surgery: Resection Margins Impact Histological Outcomes of Vocal Fold Injury

Objective—Extent of vocal fold injury impacts the nature and timing of wound healing, and voice outcomes. However, depth and extent of the lesion created to study wound healing in animal models vary across studies, likely contributing to different outcomes. Our goal was to create a surgery classification system to enable comparison of postoperative outcomes across animal vocal fold wound healing studies. Study design—Prospective, controlled animal study. Methods—Rats underwent one of three types of unilateral vocal fold surgeries classified by depth and length of resection. The surgeries were a subepithelial injury, resection of epithelium and superficial layer of the lamina propria at the midmembranous portion of the vocal fold; transmucosal injury, resection of epithelium and lamina propria; and transmuscular injury, resection of epithelium, lamina propria and superficial portion of the vocalis muscle Wound healing was evaluated histologically at various time points up to 35 days post-injury. Results—Complete healing occurred by 14 days post-surgery for subepithelial injury and by day 35 for transmucosal injury. Injury remained present at day 35 for transmuscular injury. Conclusions—Timing and completeness of healing varied by extent and depth of resection. Scarless healing occurred rapidly following subepithelial injury, while scarring was observed at five weeks after transmuscular injury. The proposed classification system may facilitate comparison of surgical outcomes across vocal fold wound healing studies

Methodology for the establishment of primary porcine vocal fold epithelial cell cultures

Objective: A current lack of methods for epithelial cell culture significantly hinders our understanding of the role of the epithelial and mucus barriers in vocal fold health and disease. Our first objective was to establish reproducible techniques for the isolation and culture of primary porcine vocal fold epithelial cells. Our second objective was to evaluate the functional significance of cell cultures using an in vitro exposure to an inflammatory cytokine. Methods: Epithelial cells were isolated from porcine vocal folds and expanded in culture. Characterization of cultures was completed by immunostaining with markers for pan-cytokeratin (epithelial cells), vimentin (stromal cells), von Willebrand factor (endothelial cell), and MUC1 and MUC4 (mucin) glycoproteins. Established epithelial cell cultures were then exposed to the inflammatory cytokine tumor necrosis factor alpha (TNF-α) for 24-hours, and transcript expression of MUC1 and MUC4 was evaluated. Results: Reproducible, porcine vocal fold epithelial cell cultures, demonstrating cobblestone appearance characteristic of the typical morphology of epithelial cell cultures were created. Cells showed positive staining for pan-cytokeratin with limited expression of vimentin and von Willebrand factor. Epithelial cells also expressed MUC1 and MUC4. TNF-α significantly increased transcript expression of MUC4. Conclusion: Here, we present the first report of successful culture of primary porcine vocal fold epithelial cells. Cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus as well as the effects of common challenges, including inflammatory cytokines, on these barriers

Successful Communication During COVID-19 for People with Speech and Hearing Challenges

Conversation with speech-language pathology faculty members, Ciara Leydon, Jamie Marotto, and Jill Douglass discussing how to communicate with individuals who have speech and hearing challenges during this time of social distancing. Hosted on Facebook by Sacred Heart University Alumni Association (32:22)

Perceptual ratings of vocal characteristics and voicing features in untreated patients with unilateral vocal fold paralysis

This study used visual analog scales to obtain perceptual ratings of features of voice production in subjects with unilateral vocal fold paralysis (UVFP), including clarity of laryngeal articulation, consistency of loudness across the utterance and the voiced/voiceless distinction. Recordings of repeated /i/, /isi/, and /izi/ from subjects diagnosed with UVFP and control subjects were randomly re-recorded, and then rated by five listeners. Significant differences in ratings (Smirnov test, p \u3c 0.01) were found between groups for aphonia , severity , clarity of articulation , overall loudness , consistency of loudness and amount of effort . Four of five raters agreed on the accuracy of /s/ or /z/ productions for only 54% of the samples from the subjects with UVFP. Voiceless and voiced cognates were equally likely to be rated as inaccurate. Results suggested that these variables were sensitive to changes in voice production resulting from paralysis, and may be useful in measuring treatment outcomes and spontaneous recovery of function. Learning outcomes: As a result of reading this manuscript the reader will (1) gain an understanding of types of perceptual scales and how to develop the set of vocal characteristics to be used in distinguishing patients with UVFP and those without, (2) learn which vocal characteristics listeners are able to use to successfully distinguish between patients with UVFP and those without and (3) understand the possible role for perceptual ratings in tracking changes in vocal characteristics in subjects over time following treatment or spontaneous recovery of function. © 2004 Elsevier Inc. All rights reserved