Faculty Bibliography

OBJECTIVE: An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent. STUDY DESIGN: In vitro. METHODS: We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-beta cascade implicated in fibroplasia in the vocal folds and other tissues. RESULTS: The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P < 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (P < 0.05). Suppression was more dose-sensitive with lipitoid. At a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a five-fold increase in lipitoid concentration. Increased number of administrations enhanced gene suppression, approximately 45% decrease between one and four administrations. Neither serum nor media type altered efficiency. CONCLUSION: Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility. LEVEL OF EVIDENCE: NA. Laryngoscope, 2016.

Therapeutic monocolonal antibodies (MAbs) are a new, rapidly growing class of medications that frequently have poorly characterized side-effect profiles. We present a patient who developed inflammatory lesions of the vocal folds in temporal relation to the initiation of alirocumab. Lesions of the vocal folds represent a previously unreported adverse effect of alirocumab therapy, making it the second MAb documented with such a side effect. The potential laryngeal effects of alirocumab specifically, and of MAbs more broadly, warrant investigation. Laryngoscope, 2016.

OBJECTIVES/HYPOTHESIS: Quantification of clinical outcomes after vocal fold (VF) interventions is challenging with current technology. High-speed digital imaging and optical coherence tomography (OCT) of excised larynges assess intact laryngeal function, but do not provide critical biomechanical information. We developed a protocol to quantify tissue properties in intact, excised VFs using dynamic nanomechanical analysis (nano-DMA) to obtain precise biomechanical properties in the micrometer scale. STUDY DESIGN: Experimental animal study. METHODS: Three pig larynges were bisected in the sagittal plane, maintaining an intact anterior commissure, and subjected to nano-DMA at nine locations with a 250-mum flat-tip punch and frequency sweep load profile (10-105 Hz, 1,000 muN peak force) across the free edge of the VF and inferiorly along the conus elasticus. RESULTS: Storage, loss, and complex moduli increased inferiorly from the free edge. Storage moduli increased from a mean of 32.3 kPa (range, 6.5-55.38 kPa) at the free edge to 46.3kPa (range, 7.4-71.6) 5 mm below the free edge, and 71.4 kPa (range, 33.7-112 kPa) 1 cm below the free edge. Comparable values were 11.6 kPa (range, 5.0-20.0 kPa), 16.7 kPa (range, 5.7-26.8 kPa), and 22.6 kPa (range, 9.7-38.0 kPa) for loss modulus, and 35.7 kPa (range, 14.4-56.4 kPa), 50.1 kPa (range, 18.7-72.8 kPa), and 75.4 kPa (range, 42.0-116.0 kPa) for complex modulus. Another larynx repeatedly frozen and thawed during technique development had similarly increased storage, loss, and complex modulus trends across locations. CONCLUSIONS: Nano-DMA of the intact hemilarynx provides a platform for quantification of biomechanical responses to a myriad of therapeutic interventions to complement data from high-speed imaging and OCT. LEVEL OF EVIDENCE: NA Laryngoscope, 2016.

OBJECTIVE: Conflicting data exist regarding false vocal fold (FVF) anatomy; it is unclear if this structure is an extension of the thyroarytenoid muscle or an independent muscle system. This confusion is amplified by diverse clinical findings in the setting of unilateral recurrent laryngeal neuropathy and presbylarynges. We sought to characterize FVF behavior in these contexts. METHODS: Laryngoscopic/stroboscopic examinations from 11 patients with unilateral recurrent laryngeal nerve paresis and 12 patients with presbylarynges were reviewed by 4 laryngologists, blinded to the goal of the study but informed of diagnosis. Variables related to FVF structure and function at rest and during phonation were rated. RESULTS: In recurrent laryngeal neuropathy, no significant association between atrophic/paretic vocal fold (VF) and FVF size was observed at rest (P = .69). During phonation, FVF compression was noted bilaterally; contralateral FVF hypertrophy was more common (P = .002). In presbylarynges, neither FVF size at rest (P = .86) nor compression during phonation (P = .37) was associated with the more atrophic VF; FVF compression/hypertrophy was common. CONCLUSIONS: Consistent with clinical dogma, FVF compression was more common contralateral to VF neuropathy. This finding, however, was inconsistent and may suggest individual variability in FVF innervation and/or morphology. Intra- and interrater reliability of these clinical findings was poor.

OBJECTIVES/HYPOTHESIS: Mesenchymal stem cells (MSCs) hold therapeutic promise for vocal fold scar, yet the precise mechanism(s) underlying tissue level changes remain unclear. We hypothesize that MSCs interact with native fibroblasts to favorably affect healing. Furthermore, we hypothesize that these interactions vary based on MSC source. METHODS: Vocal fold fibroblasts (VFFs), adipose-derived stem cells, and bone marrow-derived stem cells (BMSCs) were extracted from Sprague-Dawley rats; and a coculture model was employed culturing VFFs +/- transforming growth factor (TGF-beta1) (10 ng/mL) +/- MSCs. Monoculture MSCs were also prepared as a control. Both extracellular matrix (ECM) and components of the TGF-beta signaling pathway were analyzed via polymerase chain reaction and western blotting. RESULTS: Significantly decreased TGF-beta1 mRNA and alpha-smooth muscle actin protein was observed in VFFs in response to TGF-beta1 in the coculture with both MSCs (P < 0.05, P < 0.01). BMSCs significantly downregulated collagen I (P < 0.05), collagen III (P < 0.05), Smad3 (P < 0.01), and TGF-beta1 receptor I (P < 0.01) mRNA in VFFs. Hyaluronic synthase-1 and 2 increased in cocultured BMSCs when compared with monocultured BMSCs at baseline and in response to TGF-beta1 (P < 0.01). CONCLUSION: MSCs had a favorable effect on ECM regulation as well as suppression of TGF-beta1 signaling in VFF. Bidirectional paracrine signaling was also observed as VFFs altered ECM regulation in MSCs. These data provide insight into the regenerative effects of MSCs and provide a foundation for clinical application. LEVEL OF EVIDENCE: N/A. Laryngoscope, 2016.

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.

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.