Objectives/Hypothesis To regenerate defected recurrent laryngeal nerves (RLNs), various methods have been developed. However, no consistently effective treatments are currently available because of their insufficient functional recovery. RADA16‐I, a self‐assembling peptide used clinically as a hemostat, reportedly supports neurite outgrowth and functional synapse formation in vitro. The purpose of this study was to investigate the effect of RADA16‐I hydrogels on transected RLNs in rats. Study Design Animal experiments with controls. Methods Fifteen adult rats were divided into the following three groups: RADA16‐I (+), RADA16‐I (−), and neurectomy. A 6‐mm gap of the left RLN was bridged using an 8‐mm silicone tube in the RADA16‐I (−) and RADA16‐I (+) groups. Subsequently, RADA16‐I hydrogel was injected into the tube in the RADA16‐I (+) group. The surgical incisions were closed without any further treatment in the neurectomy group. After 8 weeks, laryngoscopy and electrophysiological and histological examinations were performed to evaluate the effect of RADA16‐I on nerve regeneration and thyroarytenoid muscle atrophy. Results Although most rats in the three groups exhibited no improvements of their vocal fold movement, partial recovery was observed in one rat in the RADA16‐I (+) group. The neurofilament‐positive areas and the number of myelinated nerves in the RADA16‐I (+) group were significantly higher than in the RADA16‐I (−) group. The area of the left thyroarytenoid muscle in the RADA16‐I (+) group was significantly larger than that of the neurectomy group. Conclusions Our results suggested that RADA16‐I hydrogel was effective for RLN regeneration. Level of Evidence NA Laryngoscope, 130:2420–2427, 2020

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. epithelial surfaces were observed using an epifluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

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Objectives Macrophages are prominent inflammatory cells in wounds, and their phenotypes are altered during wound healing. They are reported to contribute to not only inflammatory responses but also tissue remodeling. However, few studies in vocal fold biology have focused on the function of macrophages. The purpose of this study was to investigate macrophage polarization and distribution in injured murine vocal folds. Study Design Animal experiments with controls. Method Unilateral vocal fold stripping was performed on C57BL/6 mice, and larynges were harvested 1, 3, 5, 7, and 14 days postinjury. Immunohistochemical analysis of the vocal fold lamina propria was performed to detect the expression of classically activated (M1) and alternatively activated (M2) macrophage markers (inducible nitric oxide synthase [iNOS] and CD206, respectively) in F4/80+ macrophages. Results The proportion of F4/80+iNOS+ cells out of all F4/80+ cells tended to increase from day 1. F4/80+iNOS+ cell percentage tended to be high at days 1 through 7 and declined to close to a normal level by day 14. F4/80+CD206+ cell percentage tended to decrease at day 1 and then to increase the rest of the time. In the normal vocal fold, the majority of F4/80+ macrophages were only positive for CD206. F4/80+iNOS+CD206+ cells were observed at days 1 through 7. Conclusion The main population of injured sites gradually shifted from M1 to M2 marker‐positive macrophages in murine vocal folds. However, coexistence of M1 and M2 markers in the same macrophages was observed. Our results suggest that macrophage phenotypes are regulated by complex tissue‐derived signals and exhibit dynamic changes during wound healing. Level of Evidence NA Laryngoscope, 129:E135–E142, 2019

In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells