Faculty Bibliography

OBJECTIVES/HYPOTHESIS: Inhalation injury significantly increases morbidity and mortality in burn patients. Approximately one in five burn patients have acute injury to the larynx, trachea, and/or lungs-and as many as 70% have long-term laryngeal abnormalities. Although inhalation injury to the lung has been studied extensively, no models exist to study these insults to the larynx. As such, we developed an in vivo rabbit model to create precise and reproducible laryngeal burn with resultant tissue damage as a foundation for interventional studies. METHODS: Following tubeless tracheotomy, a custom temperature-control device was employed to apply heated air (70 degrees C-80 degrees C, 150 degrees C-160 degrees C, or 310 degrees C-320 degrees C) +/- smoke derived from unbleached cotton to the larynx, endoscopically, minimizing adjacent tissue damage in six rabbits. Pain, nutrition, and level of activity were monitored. Direct laryngoscopy and histological examination were performed 24 hours following insult. RESULTS: All animals survived injury with appropriate pain control; oral intake was initiated and all were adequately ventilating via tracheostomy. Burn sequelae were noted under direct visualization 24 hours after injury, and graded levels of edema and tissue damage were observed as a function of temperature. Edema obstructed true vocal fold visualization at increased temperatures. These injury patterns correlated with graded tissue damage on histology. CONCLUSION: We created an in vivo model of laryngeal burn injury employing a custom burn device resulting in graded tissue injury. This model is critical for investigation of the mechanisms underlying burn injury, and ultimately, the development and evaluation of therapies for this challenging population. LEVEL OF EVIDENCE: N/A. Laryngoscope, 2016.

The vocal folds (VFs) are exposed to a number of injurious stimuli that frequently lead to aberrant structural alterations and altered biomechanical properties that clinically manifest as voice disorders. Therapies to restore both structure and function of this delicate tissue are ideal. However, such methods have not been adequately developed. Our group and others hypothesize that tissue engineering and regenerative medicine approaches, previously described for other tissue systems, hold significant promise for the VFs. In the current review, we explore the concept of tissue engineering as it relates to the VFs as well as recent studies employing both naturally- and synthetically-derived biomaterials, including those from laryngeal and non-laryngeal sources, in combination with stem cells for a tissue-engineered approach to VF repair.

OBJECTIVE: To determine the prevalence and also accuracy of the laryngopharyngeal reflux (LPR) referring diagnosis and to determine the most useful clinical tool in arriving at the final diagnosis in a tertiary laryngology practice. STUDY DESIGN: Case series with planned data collection. SETTING: Six tertiary academic laryngology practices. SUBJECTS AND METHODS: We collected referring diagnosis and demographic information, including age, sex, ethnicity, referring physician, and whether or not patients had prior flexible laryngoscopy for 1077 patients presenting with laryngologic complaints from January 2010 and June 2013. Final diagnosis after the referred laryngologist's examination and the key diagnostic test used was then recorded. RESULTS: Of 1077 patients, 132 had a singular referring diagnosis of LPR. Only 47 of 132 patients (35.6%) had LPR confirmed on final primary diagnosis. Transnasal flexible laryngoscopy confirmed this in 27 of 47 (57.4%) patients. Eighty-five of 132 (64.4%) had a different final diagnosis than LPR. Sixty-five of 85 (76.5%) of these alternative pathologies were diagnosed with the aid of laryngeal stroboscopy. CONCLUSIONS: LPR appears to be an overused diagnosis for laryngologic complaints. For patients who have already had transnasal flexible laryngoscopic exams prior to their referral, laryngeal stroboscopy is the key diagnostic tool in arriving at the correct diagnosis.

The human vocal folds are complex structures made up of distinct layers that vary in cellular and extracellular composition. The mechanical properties of vocal fold tissue are fundamental to the study of both the acoustics and biomechanics of voice production. To date, quantitative methods have been applied to characterize the vocal fold tissue in both normal and pathologic conditions. This review describes, summarizes, and discusses the most commonly employed methods for vocal fold biomechanical testing. Force-elongation, torsional parallel plate rheometry, simple-shear parallel plate rheometry, linear skin rheometry, and indentation are the most frequently employed biomechanical tests for vocal fold tissues and each provide material properties data that can be used to compare native tissue to diseased or treated tissue. Force-elongation testing is clinically useful, as it allows for functional unit testing, while rheometry provides physiologically relevant shear data, and nanoindentation permits micrometer scale testing across different areas of the vocal fold as well as whole organ testing. Thoughtful selection of the testing technique during experimental design to evaluate a hypothesis is critical to optimize biomechanical testing of vocal fold tissues.

We report a case of unilateral vocal fold immobility in a 57-year-old woman that occurred subsequent to a choking episode, which she resolved by removing impacted food with a finger sweep. Other than the unilateral immobility, no abnormality of the laryngeal mucosa or framework was detected on physical examination, laryngoscopy, and computed tomography. Weeks later, the patient underwent an unrelated surgical procedure necessitating laryngeal mask airway ventilation. When she emerged from the procedure, she noted full resolution of her voice symptoms. Office laryngoscopy confirmed the full return of vocal fold function. We discuss possible explanations for the return of function in the context of this unusual onset and resolution. We also review the literature regarding unilateral vocal fold immobility, including its etiology, presentation, workup, and treatment.

OBJECTIVES/HYPOTHESIS: To investigate the role of Smad3 as a regulator of transforming growth factor (TGF)-beta1-mediated cell activities associated with fibrosis in normal human vocal fold fibroblasts. We also sought to confirm the temporal stability of Smad3 knockdown via small inhibitor ribonucleic acid (siRNA). Vocal fold fibroblasts were employed to determine the effects of Smad3 knockdown on TGF-beta1-mediated migration and contraction, as well as regulation of connective tissue growth factor (CTGF). We hypothesized that Smad3 is an ideal candidate for therapeutic manipulation in vivo based on its role in fibrosis. STUDY DESIGN: In vitro. METHODS: Knockdown of Smad3 via siRNA was performed in our normal human vocal fold cell line. Three-dimensional collagen gel contraction and scratch assays were employed to determine the role of Smad3 on TGF-beta1-mediated contraction and migration, respectively. The role Smad3 in the induction of CTGF was characterized via sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of Smad3 signaling on Smad7 messenger (m)RNA and protein were also quantified. RESULTS: Smad3 knockdown was temporally-stable up to 72 hours (P < 0.001), diminished TGF-beta1-mediated collagen gel contraction and migration, and blunted induction of CTGF, but it had no effect on TGF-beta1-mediated Smad7 mRNA or protein induction. CONCLUSION: Transforming growth factor-beta1 stimulated profibrotic cell activities in our cell line and these actions were largely reduced with Smad3 knockdown. These data provide continued support for therapeutic targeting of Smad3 for vocal fold fibrosis because it appears to regulate the fibrotic phenotype. LEVEL OF EVIDENCE: N/A. Laryngoscope, 2015.

OBJECTIVES/HYPOTHESIS: To optimize decellularization of porcine vocal folds (VF) and quantify human bone marrow-derived mesenchymal stem cell (BM-MSC) interactions with this matrix to provide a foundation for regenerative approaches to VF repair. STUDY DESIGN AND METHODS: Vocal folds were dissected from porcine larynges and three decellularization protocols were compared, each consisting of washes and mechanical agitations with different combinations of reagents. DNA content was analyzed via Quant-iT Picogreen assay and hematoxylin and eosin staining. Bone marrow-derived MSCs were then seeded onto the decellularized VF matrices. Morphology, metabolic activity, DNA content, and gene expression were assessed using LIVE/DEAD Cell Viability, alamarBlue Cell Viability Assay, Quant-iT Picogreen assay, and, respectively. RESULTS: The most successful decellularization protocol removed 95% DNA content within 1 day, compared to several days required for previously described protocols. Histology confirmed the retention of extracellular matrix (ECM) and its components, including glycosaminoglycans, collagen, and fibrin, while void of nuclear/cellular content. Decellularized scaffolds were then seeded with BM-MSCs. Similar DNA quantities were observed after 24 hours of seeding within the VF-ECM scaffold when compared to cells on tissue culture plastic (TCP). LIVE/DEAD staining of the seeded VF-ECM confirmed excellent cell viability, and the metabolic activity of BM-MSCs increased significantly on VF-ECM compared to TCP. Endoglin gene expression decreased, suggestive of differentiation. CONCLUSION: Porcine VFs can be efficiently decellularized within 5 hours using a combination of sodium deoxycholate and peracetic acid. Decellularized VF-ECM supported attachment and growth of human BM-MSCs, with evidence of differentiation. LEVEL OF EVIDENCE: N/A Laryngoscope, 2015.

Purpose: Dynamic MRI has been used to examine swallowing in sagittal and axial planes with good temporal resolution. However, few studies have examined temporal measures and none have compared upright vs. supine swallowing in both positions with the MBSImP. We report physiologic variables and swallow safety upright and supine with MBSImP and Penetration/Aspiration scales in healthy subjects. Method(s): Twenty healthy subjects (mean = 28 years) underwent VFS study upright and supine with a C-Arm. Subjects were viewed in the sagittal plane and swallowed 5 mL liquid and pudding barium. Pen/Asp scores and 14 MBSImP variables were analyzed (overall Impression score) in both positions, with means and ranges across subjects upright and supine. Paired t-tests compared MBSImP scores upright vs. supine. Level of significance: p<.05. Result(s): All subjects tolerated the supine position while swallowing. Mean MBSImP scores were WNL across subjects for both positions, except Component 6, Initiation of the Pharyngeal Swallow (Table 1). Nosignificant differences were found for MBSImP scores upright vs. supine. Although the range of Pen/Asp scores was higher for supine, 3 subjects penetrated upright only, 1 penetrated supine only, and 1 aspirated on 1 supine swallow. Pearson coefficients for inter/intra-judge reliability:0.94, 0.91. Conclusions (Including Clinical Relevance): Physiologic measurse revealed a pharyngeal delay in both positions with scores not significantly different. Although Pen/Asp range was higher in supine, more subjects penetrated upright. Future research should examine swallow physiology in both positions in dysphagic and older healthy subjects. (Table Presented)

Purpose: Age-related loss of muscle bulk and strength has been documented in the tongue and geniohyoid. Our goal was to explore this phenomenon in the pharynx, specifically by measuring pharyngeal wall thickness (PWT) and pharyngeal lumen area (PLA) in a sample of young vs older women. Method(s): MRI scans of the neck were retrospectively reviewed from 60 women equally stratified by 3 age groups (20s, 60s, 70+). Exclusion criteria included dysphagia, c-spine surgery, neurological illness, head and neck malignancy and obstructive sleep apnea. Three de-identified axial slices were extracted per scan for randomized, blinded analysis: at the levels of the anterior inferior border of C2 and C3, and at the pit of the vallecula (Vpit). Pixel-based measures of PWT and PLA were completed using ImageJ and converted to metric units using the calibration markers on the original images. Measures of PWT and PLA (at three levels) were compared between age groups with one-way ANOVAs using Sidak adjustments for post-hoc pairwise comparisons. Result(s): A significant main effect of age was observed for all variables whereby PWT decreases and PLA increases with advancing age (Table 1). Pairwise comparisons revealed significant differences between 20s vs 70+ for all variables and 20s vs 60s for all variables except PWT and PLA at C2. Effect sizes ranged from 0.56-1.34. Conclusions (Including Clinical Relevance): Consistent with the existing sacropenia literature, the pharyngeal muscles appear to atrophy with age and consequently, PLA increases as well. Future work should explore the relationship between pharyngeal muscle size/ atrophy and functional swallowing outcomes. (Table Presented)