Effects of fabrication parameters on viscoelastic shear modulus of 2,3‐dialdehydecellulose membranes—Potential scaffolds for vocal fold lamina propria tissue engineering

The lamina propria within the vocal fold (VF) is a complex multi-layered tissue that increases in stiffness from the superficial to deep layer, where this characteristic is crucial for VF sound production. Tissue engineered scaffolds designed for VF repair must mimic the biophysical nature of the native vocal fold and promote cell viability, cell spreading, and vibration with air flow. In this study, we present a unique tri-layered, partially-degradable hydrogel scaffold that mimics the multi-layered structure of the VF lamina propria. Using thiol-norbornene photochemistry, tri-layered hydrogel scaffolds were fabricated via layer-by-layer stacking with increasing polymer concentration from the top to middle to deep layer. Mechanical analysis confirmed hydrogel modulus increased with increasing polymer concentration. Partially-degradable hydrogels promoted high cell viability and cell spreading in 3D as assessed via live/dead and cytoskeleton staining, respectively. Importantly, partially-degradable hydrogels maintained some degree of the 3D polymer network following protease exposure, while still enabling encapsulated cells to remodel their local environment via protease secretion. Finally, the tri-layered hydrogel scaffold successfully vibrated and produced sound in proof-of-concept air flow studies. This work represents a critical first step towards the design of a multi-layered, hydrogel scaffold for vocal fold tissue engineering.

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Cogels of Hyaluronic Acid and Acellular Matrix for Cultivation of Adipose-Derived Stem Cells: Potential Application for Vocal Fold Tissue Engineering

BioMed Research International ◽

10.1155/2016/6584054 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Dongyan Huang ◽

Rongguang Wang ◽

Shiming Yang

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Extracellular Matrix ◽

Hyaluronic Acid ◽

Lamina Propria ◽

Vocal Fold ◽

Adipose Derived Stem Cells ◽

Acellular Matrix ◽

Decellularized Extracellular Matrix ◽

Superficial Lamina

Stem cells based tissue engineering has been one of the potential promising therapies in the research on the repair of tissue diseases including the vocal fold. Decellularized extracellular matrix (DCM) as a promising scaffold has be used widely in tissue engineering; however, it remained to be an important issue in vocal fold regeneration. Here, we applied the hydrogels (hyaluronic acid [HA], HA-collagen [HA-Col], and HA-DCM) to determine the effects of hydrogel on the growth and differentiation of human adipose-derived stem cells (hADSCs) into superficial lamina propria fibroblasts. hADSCs were isolated and characterized by fluorescence-activated cell sorting. The results indicated that HA-DCM hydrogel enhanced cell proliferation and prolonged cell morphology significantly compared to HA and HA-Col hydrogel. Importantly, the differentiation of hADSCs into fibroblasts was also promoted by cogels of HA-Col and HA-DCM significantly. The differentiation of hADSCs towards superficial lamina propria fibroblasts was accelerated by the secretion of HGF, IL-8, and VEGF, the decorin and elastin expression, and the synthesis of chondroitin sulfate significantly. Therefore, the cogel of HA-DCM hydrogel was shown to be outstanding in apparent stimulation of hADSCs proliferation and differentiation to vocal fold fibroblasts through secretion of important growth factors and synthesis of extracellular matrix.

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Reinke's Edema: Phonatory Mechanisms and Management Strategies

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348949710600701 ◽

1997 ◽

Vol 106 (7) ◽

pp. 533-543 ◽

Cited By ~ 68

Author(s):

Steven M. Zeitels ◽

Glenn W. Bunting ◽

Robert E. Hillman ◽

Traci Vaughn

Keyword(s):

Lamina Propria ◽

Fundamental Frequency ◽

Vocal Fold ◽

Management Strategies ◽

Vocal Folds ◽

Vocal Fold Vibration ◽

Reinke’S Edema ◽

Subglottal Pressure ◽

Almost All ◽

Superficial Lamina

Reinke's edema (RE) has been associated typically with smoking and sometimes with vocal abuse, but aspects of the pathophysiology of RE remain unclear. To gain new insights into phonatory mechanisms associated with RE pathophysiology, weused an integrated battery of objective vocal function tests to analyze 20 patients (19 women) who underwent phonomicrosurgical resection. Preoperative stroboscopic examinations demonstrated that the superficial lamina propria is distended primarily on the superior vocal fold surface. Acoustically, these individuals have an abnormally low average speaking fundamental frequency (123 Hz), and they generate abnormally high average subglottal pressures (9.7 cm H20). The presence of elevated aerodynamic driving pressures reflects difficulties in producing vocal fold vibration that are most likely the result of mass loading associated with RE, and possibly vocal hyperfunction. Furthermore, it is hypothesized that in the environment of chronic glottal mucositis secondary to smoking and reflux, the cephalad force on the vocal folds by the subglottal driving pressure contributes to the superior distention of the superficial lamina propria. Surgical reduction of the volume of the superficial lamina propria resulted in a significant elevation in fundamental frequency (154 Hz) and improvement in perturbation measures. In almost all instances, both the clinician and the patient perceived the voice as improved. However, these patients continued to generate elevated subglottal pressure (probably a sign of persistent hyperfunction) that was accompanied by visually observed supraglottal strain despite the normalsized vocal folds. This finding suggests that persistent hyperfunctional vocal behaviors may contribute to postsurgical RE recurrence if therapeutic strategies are not instituted to modify such behavior.

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Immunocytochemical Study of Proteoglycans in Vocal Folds

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348949610500102 ◽

1996 ◽

Vol 105 (1) ◽

pp. 6-11 ◽

Cited By ~ 72

Author(s):

Agnieszka S. Pawlak ◽

Elizabeth Hammond ◽

Thomas Hammond ◽

Steven D. Gray

Keyword(s):

Chondroitin Sulfate ◽

Lamina Propria ◽

Vocal Fold ◽

Keratan Sulfate ◽

Vocal Folds ◽

Basement Membrane Zone ◽

Immunocytochemical Study ◽

Frozen Sections ◽

Intense Staining ◽

Immunocytochemical Techniques

We evaluated the proteoglycan composition of normal vocal folds using immunocytochemical techniques. Frozen sections of 14 normal cadaveric vocal folds were obtained within 12 hours of death and sectioned immediately. Vocal fold sections were stained with antibodies against keratan sulfate, chondroitin sulfate, heparan sulfate proteoglycan (HSPG), decorin, and hyaluronate receptor. We found that the lamina propria has diffuse staining of fibrillar components with keratan sulfate and decorin. Intense staining was observed in the vocal ligament area with keratan sulfate. The HSPG was localized to the basement membrane zone. Chondroitin sulfate, HSPG, and hyaluronate receptor were detected in the cytoplasm of interstitial cells with immunocytochemical characteristics of macrophages. The keratan sulfate distribution suggests that fibromodulin may be significant in normal vocal folds. Production of HSPG and probably versican occurs in macrophages and fibroblasts in the lamina propria.

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Collagen composite hydrogels for vocal fold lamina propria restoration

Biomaterials ◽

10.1016/j.biomaterials.2005.07.022 ◽

2006 ◽

Vol 27 (7) ◽

pp. 1104-1109 ◽

Cited By ~ 95

Author(s):

Mariah S. Hahn ◽

Benjamin A. Teply ◽

Molly M. Stevens ◽

Steven M. Zeitels ◽

Robert Langer

Keyword(s):

Lamina Propria ◽

Vocal Fold ◽

Composite Hydrogels

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Histological Structure of Vocal Fold Lamina Propria in Foetal Larynx

Acta Oto-Laryngologica ◽

10.3109/00016488409132912 ◽

1984 ◽

Vol 97 (5-6) ◽

pp. 403-406 ◽

Cited By ~ 16

Author(s):

Radovan Suboti ◽

Santa Večerina ◽

Zvonimir Krajina ◽

Minoru Hirano ◽

Shigejiro Kurita

Keyword(s):

Lamina Propria ◽

Vocal Fold ◽

Histological Structure

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A method for identification of vocal fold lamina propria fibroblasts in culture

Otolaryngology ◽

10.1016/j.otohns.2008.09.009 ◽

2008 ◽

Vol 139 (6) ◽

pp. 816-822 ◽

Cited By ~ 29

Author(s):

Susan L. Thibeault ◽

Wenhua Li ◽

Stephanie Bartley

Keyword(s):

Lamina Propria ◽

Cell Lines ◽

Von Willebrand Factor ◽

Vocal Fold ◽

Fibroblast Cell ◽

Cytokeratin 19 ◽

Von Willebrand ◽

Willebrand Factor ◽

Area Of Study

Objective Vocal fold biology research is emerging as a vital area of study in laryngology. One impediment is the lack of both commercially available vocal fold lamina propria fibroblasts and a constitutively expressed specific marker for fibroblasts. We present an in vitro technique that allows for identification of fibroblasts by ruling out the possibility of the cells belonging to other lineages that are found in vocal fold tissue. Study Design An in vitro study. Methods Two primary vocal fold fibroblast cell lines and one immortalized vocal fold fibroblast cell line were cultured. Immunohistologic staining for α-actinin, cytokeratin 19, and von Willebrand factor was completed for the three fibroblast lines in addition to skeletal, endothelial, and epithelial cell lines. Cell type was differentiated by positive staining for α-actinin, cytokeratin 19, and von Willebrand factor. Results Fibroblast cultures did not express α-actinin, cytokeratin 19, and von Willebrand factor, whereas skeletal muscle, endothelial, and epithelial cultured cells expressed each respectively. Conclusions This simple rule-out methodology for fibroblast confirmation is an important step when establishing cell culture, and it establishes sound internal validity particularly in the early stages of this emerging area of study.

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