Tissue engineering of the larynx: A contemporary review.

OBJECTIVE: Tissue engineering has been a topic of extensive research in recent years and has been applied to the regeneration and restoration of many organs including the larynx. Currently, research investigating tissue engineering of the larynx is either ongoing or in the preclinical trial stage. METHODS: A literature search was performed on the Advanced search field of PubMed using the keywords: "(laryncheal tissue engineering) AND (cartilage regeneration OR scaffolds OR stem cells OR biomolecules)." After applying the selection criteria, 65 articles were included in the study. RESULTS: The present review focuses on the rapidly expanding field of tissue-engineered larynx, which aims to provide stem cell-based scaffolds combined with biological active factors such as growth factors for larynx reconstruction and regeneration. The trend in recent studies is to use new techniques for scaffold construction, such as 3D printing, are developed. All of these strategies have been instrumental in guiding optimization of the tissue-engineered larynx, leading to a level of clinical induction beyond the in vivo animal experimental phase. CONCLUSIONS: This review summarizes the current progress and outlines the necessary basic components of regenerative laryngeal medicine in preclinical fields. Finally, it considers the design of scaffolds, support of growth factors, and cell therapies toward potential clinical application.

M2 Macrophages Promote Collagen Expression and Synthesis in Laryngotracheal Stenosis Fibroblasts.

OBJECTIVE: Macrophages exhibit distinct phenotypes and are dysregulated in a model of iatrogenic laryngotracheal stenosis (iLTS). Increased populations of alternatively activated or M2 macrophages have been demonstrated. However, the role of these macrophages is unknown. The aims of this study are: 1) define the macrophage population in iLTS in the context of classically activated or M1 and M2 macrophage phenotypes, and 2) characterize the effect of monocyte-derived M1 and M2 macrophages on normal airway and LTS-derived fibroblasts (FBs) in vitro. STUDY DESIGN: Comparative analysis; in vitro controlled study. METHODS: Immunohistochemical analysis of human iLTS and control specimens was performed to define the macrophage population. In vitro, M1, and M2 macrophages were polarized using M-CSF + Interferon-gamma and lipopolysaccharide or Interleukin-4, respectively. FBs isolated from laryngotracheal scar (LTS-FBs) and normal tracheal airway (NA-FBs) in eight patients with iLTS were cocultured with polarized macrophages. Fibrosis gene expression, soluble collagen production, and proliferation were assessed. RESULTS: Immunohistochemical analysis revealed increased CD11b + cells (macrophage marker) in laryngotracheal scar specimens (18.3% vs. 8.5%, P = .03) and predominant CD206 (M2) costaining versus CD86 (M1) (51.5% vs. 9.8%, n = 10, P = .001). In vitro, NA-FBs cultured with M2 macrophages demonstrated a 2.41-fold increase in collagen-1 expression (P = .05, n = 8) and an increase in soluble collagen (9.98 vs. 8.875, mean difference: 1.11 95%, confidence interval 0.024-2.192, n = 8, P = .015). CONCLUSION: Increased populations of CD11b cells are present in iLTS specimens and are predominantly CD206+, indicating an M2 phenotype. In vitro, M2 macrophages promoted collagen expression in airway FBs. Targeting macrophages may represent a therapeutic strategy for attenuating fibrosis in iLTS. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E346-E353, 2021.

RNA Sequencing Reveals Cancer-Associated Changes in Laryngeal Cells Exposed to Non-Acid Pepsin.

OBJECTIVE: Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN: In vitro translational. METHODS: Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS: Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. CONCLUSIONS: A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:121-129, 2021.

The role of pepsin in epithelia-mesenchymal transition in idiopathic subglottic stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is commonly characterized by laryngeal fibrosis thought to arise by epithelia-mesenchymal transition (EMT) induced by chronic inflammation. Pepsin is a potent inducer of inflammation in the airways during chronic laryngopharyngeal reflux and has been observed in the subglottic mucosa of patients with iSGS, absent in normal mucosa. The aim of this study was to examine the effect of pepsin on mechanisms of EMT in laryngeal cells with implications for iSGS. STUDY DESIGN: In vitro translational research study. METHODS: Human laryngeal epithelial cell cultures were exposed to 0.1 mg/mL or 1.0 mg/mL pepsin at pH7 for 24 and 48 hours, or media pH5 ± 0.1 mg/mL pepsin for 10 minutes and harvested after 24 and 48 hours. EMT marker expression was measured by qPCR and enzyme-linked immunosorbent assays. Wound-healing scratch assay was performed on immortalized human vocal fold fibroblasts pretreated with media pH5 ± 0.1 mg/mL pepsin (10 minutes) or continuously treated with media pH7 ± 0.1 to 1 mg/mL pepsin for 24 hours. RESULTS: Pepsin yielded no effect on MMP1, MMP9, FN1, COL1A1, HAS2, or CDH1 gene expression or matrix metalloproteinase-9 or fibronectin protein expression, either alone or in the presence of weak acid. Pepsin and/or acid produced no effect on fibroblast migration. CONCLUSION: Whereas pepsin has been shown to be present in the subglottic mucosa of patients with iSGS, this in vitro acute exposure investigation does not provide evidence of a direct causal role for development of fibrosis in subglottic epithelial cell cultures. LEVELS OF EVIDENCE: NA. Laryngoscope, 130:154-158, 2020.

Assessment of cancer stem cell marker expression in primary head and neck squamous cell carcinoma shows prognostic value for aldehyde dehydrogenase (ALDH1A1).

Cancer stem cells (CSCs) play a key role in carcinogenesis and progression of head and neck squamous cell carcinomas (HNSCC). The most common markers indicating for CSCs are: CD44, CD24, CD133, ALDH1A1. Our objective was to evaluate the prognostic potential of CSC markers in HNSCC. The study included 49 patients treated for primary HNSCC, 11 patients with upper respiratory tract epithelial dysplasia and 12 subjects with the normal pharyngeal mucosa as a control group. The frequency and expression levels of the four CSC markers were assessed by immunohistochemistry. Univariate and multivariate analyses were used to correlate CSC expression levels with tumor stage, lymph node metastases or overall survival (OS). CD44, CD24, CD133, ALDH1A1 were widely expressed in tumors, whereas CD44 was found to be higher in cancer tissue (P = 0.001). ALDH1A1 expression levels were found to be significantly higher in T3-T4 tumors vs. T1-T2 tumors (P = 0.05). Lymph node metastases had significantly higher expression levels of CD24 (P = 0.01) and CD133 (P < 0.05) than primary tumors. Multifactorial analysis revealed that overall survival (OS) for patients with ALDH1A1 negative tumors was 5.25 times higher than for patients with ALDH1A1 positive (ALDH1A1+) tumors (P = 0.01). On univariate and multivariate analysis, only ALDH1A1 positivity had a significant effect on OS of HNSCC patients (HR = 2.47 for P = 0.02). Immunohistochemistry-based assessments of CSC marker expression in HNSCC has significant predictive implications for patients with HNSCC. The frequency of CSCs in the tumor, specifically of ALDH1A1+ cells correlated with five-year OS in these patients.

Creation of Laryngeal Grafts from Primary Human Cells and Decellularized Laryngeal Scaffolds.

Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration.

Optimized Generation of Primary Human Epithelial Cells from Larynx and Hypopharynx: A Site-Specific Epithelial Model for Reflux Research.

Laryngopharyngeal reflux (LPR) induces a differential damage effect on several anatomic sites within the larynx and hypopharynx; therefore, an in vitro model is needed for each anatomic site. This study aimed to establish a primary culture method for human laryngeal and hypopharyngeal epithelial cells derived from multiple anatomic sites. Surgical mucosa specimens were treated with a two-step enzymatic strategy to establish a primary culture. Of the 46 samples, primary cultivation was achieved successfully with 36 samples, and the positive ratio was 78.3%. In addition, flow cytometry revealed that these primary cells were epithelial cells with a purity of 94.9%. The proliferative ability was confirmed by positive staining for Ki-67. Laryngeal and hypopharyngeal epithelial cells from multiple sites exhibited similar epithelial morphology and positive cytokeratin expression. These cells can be cultured to passage 4. In summary, we successfully established the in vitro epithelial model of larynx and hypopharynx subsites, which may potentially be used as a platform for reflux research, especially for site-specific damage effect.

Sensory Innervation of the Human Soft Palate.

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc.

Extrinsic and Intrinsic Apoptotic Responses Induced by Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Aqueous Extract against a Larynx Carcinoma Cell Line.

Cumulative evidence from research studies has shown that the shiitake culinary-medicinal mushroom, Lentinus edodes, is an excellent source of natural antitumor agents and is capable of inhibiting cancer cell growth. However, the cell signaling pathway that leads tumor cells to apoptosis is not well understood because many chemical compounds may be acting. This study investigated the chemopreventive effects of an L. edodes aqueous extract on human HEp-2 epithelial larynx carcinoma cells and normal human MRC-5 lung fibroblasts by identifying proliferative and apoptotic pathways. The chemical characterization of the dry powder was assessed by high-performance liquid chromatography. Antiproliferative and proapoptotic effects induced by the extract were evaluated by assessing proliferative markers, cell sorting through flow cytometry, and expression levels of apoptotic proteins with Western blotting. The results suggest that inhibition of cell proliferation was more prominent in HEp-2 than in MRC-5 cells. Cell death analysis showed the appearance of cell populations in the sub-G1 phase, with late apoptotic signal increased in a dose-dependent manner. In addition, the aqueous extract induced depolarization of mitochondria, activating the generation of intracellular reactive oxygen species in HEp-2 cells. These observations suggest that L. edodes extract may exert a chemopreventive effect, regulating mitotic induction of apoptogenic signals. These findings highlight the mushroom's pharmacological potential in cancer treatment.

Immunohistochemical characterization of brush cells in the rat larynx.

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, ß2-subunit (PLCß2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.

A Method to Administer Agents to the Larynx in an Awake Large Animal.

Purpose: This research note describes an adapted experimental methodology to administer an exogenous agent to the larynx and upper airway of awake animals. The exogenous agent could be a perturbation. In the current study, the agent was isotonic saline. Isotonic saline was selected because it is safe, of similar composition to extracellular fluid, and used in voice studies. The described approach allowed large animals such as pigs to be comfortably restrained without chemical sedation or anesthesia for extended periods while receiving the agent. Method: Six Sinclair pigs were successfully trained with positive reinforcement to voluntarily enter and then be restrained in a Panepinto Sling. Once restrained, the pigs accepted a nose cone that delivered nebulized isotonic saline. This procedure was repeated 3 times per day for 20 days. At the end of the study, the larynx and airway tissues were excised and examined using histology and transmission electron microscopy. Results: Pathology related to the procedure (i.e., nebulized inhaled isotonic saline or stress) was not identified in any examined tissues. Conclusions: This methodology allowed for repeated application of exogenous agents to awake, unstressed animals. This method can be used repeatedly in the laboratory to test various therapeutics for safety, toxicity, and dosage. Future studies will specifically manipulate the type of agent to further our understanding of laryngeal pathobiology.

Comparative characteristics of laryngeal-resident mesenchymal stromal cell populations isolated from distinct sites in the rat larynx.

BACKGROUND: Although tissue-resident mesenchymal stromal cells (MSCs) in the larynx have been described, their distinct characteristics and roles have not been thoroughly explored. Therefore, we investigated stem cell characteristics and regenerative potentials of single clonal populations isolated from rat epiglottic mucosa (EM), lamina propria (LP), and macula flava (MF) to determine whether they comprised laryngeal tissue-resident stem cells. METHODS: Single clonal laryngeal cells were isolated following microdissection of the EM, LP, and MF from the rat larynx. Several clonal populations from the three laryngeal subsites were selected and expanded in vitro. We compared the stem cell characteristics of self-renewal and differentiation potential, as well as the cell surface phenotypes and gene expression profiles, of laryngeal MSC-like cells to that of bone marrow MSCs (BM-MSCs). We also investigated the regenerative potential of the laryngeal cells in a radiation-induced laryngeal injury animal model. RESULTS: Self-renewing, clonal cell populations were obtained from rat EM, LP, and MF. EM-derived and LP-derived clonal cells had fibroblast-like features, while MF-resident clonal cells had stellate cell morphology and lipid droplets containing vitamin A. All laryngeal clonal cell populations had MSC-like cell surface marker expression (CD29, CD44, CD73, and CD90) and the potential to differentiate into bone and cartilage cell lineages; EM-derived and MF-derived cells, but not LP-derived cells, were also able to differentiate into adipocytes. Clonal cells isolated from the laryngeal subsites exhibited differential extracellular matrix-related gene expression. We found that the mesenchymal and stellate cell-related genes desmin and nestin were enriched in laryngeal MSC-like cells relative to BM-MSCs (P < 0.001). Growth differentiation factor 3 (GDF3) and glial fibrillary acidic protein (GFAP) transcript and protein levels were higher in MF-derived cells than in other laryngeal populations (P < 0.001). At 4 weeks after transplantation, laryngeal MF-derived and EM-derived cells contributed to laryngeal epithelial and/or glandular regeneration in response to radiation injury. CONCLUSIONS: These results suggest that cell populations with MSC characteristics reside in the EM, LP, and MF of the larynx. Laryngeal MSC-like cells contribute to regeneration of the larynx following injury; further investigation is needed to clarify the differential roles of the populations in laryngeal tissue regeneration, as well as the clinical implications for the treatment of laryngeal disease.

An in vitro scaffold-free epithelial-fibroblast coculture model for the larynx.

OBJECTIVES/HYPOTHESIS: Physiologically 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 TGFß1. STUDY DESIGN: In vitro. METHODS: Cell 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 ß1 (TGFß1) 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. RESULTS: Fibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFß1-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. CONCLUSIONS: These 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 EVIDENCE: NA Laryngoscope, 127:E185-E192, 2017.

Identification of cholinergic chemosensory cells in mouse tracheal and laryngeal glandular ducts.

Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect "bitter" substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel = TRPM5, phospholipase C(ß2)), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways.

Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging.

The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n=10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert's resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging.

Establishment of an immortalized laryngeal posterior commissure cell line as a tool for reflux research.

OBJECTIVES/HYPOTHESIS: Laryngopharyngeal reflux (LPR) has been implicated as a promoter of laryngeal cancer. Within the larynx, the posterior commissure (PC) is the region that usually comes into direct contact with refluxed materials. Specific laryngeal cell lines useful for in vitro studies are not widely available, and noncancer-derived PC laryngeal cell line has not yet been described. STUDY DESIGN: Experimental study. METHODS: Specimens of squamous epithelium from the PC of the larynx were collected from patients without a history or evidence of laryngeal inflammatory or neoplastic diseases. Harvested tissue was cultured and then immortalized by transduction with human papillomavirus E6/E7-encoding lentivirus. PC primary and transformed cells were characterized by light microscopy and immunohistochemistry. RESULTS: Primary cultures established from PC contained < 5% fibroblasts and displayed normal epithelial cell morphology and cytokeratin expression. These cells survived nine passages in culture. Following lentiviral-mediated immortalization, cells retained normal squamous epithelial morphology and survived > 20 passages in culture. Methods were optimized for culture of PC laryngeal epithelial cells, resulting in 90% success rate of culture. CONCLUSION: A novel immortalized PC laryngeal epithelial cell line has been established. This cell line provides a unique tool for investigating the mechanism of LPR in the development and progression of laryngeal cancer. LEVEL OF EVIDENCE: N/A.

A flexible wide-field FLIM endoscope utilising blue excitation light for label-free contrast of tissue.

Fluorescence lifetime imaging (FLIM) has previously been shown to provide contrast between normal and diseased tissue. Here we present progress towards clinical and preclinical FLIM endoscopy of tissue autofluorescence, demonstrating a flexible wide-field endoscope that utilised a low average power blue picosecond laser diode excitation source and was able to acquire ∼mm-scale spatial maps of autofluorescence lifetimes from fresh ex vivo diseased human larynx biopsies in ∼8 seconds using an average excitation power of ∼0.5 mW at the specimen. To illustrate its potential for FLIM at higher acquisition rates, a higher power mode-locked frequency doubled Ti:Sapphire laser was used to demonstrate FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen.

Automated labeling of cancer textures in larynx histopathology slides using quasi-supervised learning.

OBJECTIVE: To evaluate the performance of a quasi-supervised statistical learning algorithm, operating on datasets having normal and neoplastic tissues, to identify larynx squamous cell carcinomas. Furthermore, cancer texture separability measures against normal tissues are to be developed and compared either for colorectal or larynx tissues. STUDY DESIGN: Light microscopic digital images from histopathological sections were obtained from laryngectomy materials including squamous cell carcinoma and nonneoplastic regions. The texture features were calculated by using co-occurrence matrices and local histograms. The texture features were input to the quasi-supervised learning algorithm. RESULTS: Larynx regions containing squamous cell carcinomas were accurately identified, having false and true positive rates up to 21% and 87%, respectively. CONCLUSION: Larynx squamous cell carcinoma versus normal tissue texture separability measures were higher than colorectal adenocarcinoma versus normal textures for the colorectal database. Furthermore, the resultant labeling performances for all larynx datasets are higher than or equal to that of colorectal datasets. The results in larynx datasets, in comparison with the former colorectal study, suggested that quasi-supervised texture classification is to be a helpful method in histopathological image classification and analysis.

Development of the rat larynx: a histological study.

OBJECTIVES/HYPOTHESIS: To evaluate and describe the cartilaginous and muscular development of the rat larynx. STUDY DESIGN: Histologic evaluation. METHODS: The larynges of Sprague Dawley rats of embryonic day (E) 13, 15, 17, 19, 21, postnatal day 0, 14, and adult of 250 gm were collected. Four larynges of each age were harvested, cut into 15-µm serial sections, stained with hematoxylin and eosin, and evaluated under light microscopy. Representative digital images were recorded and evaluated at the preglottic (supraglottic in humans), glottic, and postglottic (subglottic in humans) levels. RESULTS: Brachial arches were observed at E13. At E17, immature structures of the larynx, including skeletal muscle, cartilage, and the lumen were identifiable. Chondrification and muscle formation were clearly seen by E19. The muscular and cartilagenous components of the larynx were well established by E21. During the span between birth and adult maturation, the size of the larynx increased from a height of 1.10 mm to 2.90 mm, and from a width of 1.80 mm to 5.40 mm, and from a length of 1.38 mm to 4.77 mm in the stained section. Although developed at E21, the laryngeal structures continued to grow by approximately 30%. CONCLUSION: Rat laryngeal development parallels that in mice and humans. In the rat, at E17 immature structures of the larynx are identifiable, they are well developed at birth and grow by approximately 30% into adulthood. Understanding the chronology and morphology of the embryogenesis of the rat laryngeal musculature is essential and will allow for further evaluation of the embryologic innervation of these muscles.