Functional testing of a tissue-engineered vocal fold cover replacement

ABSTRACT OBJECTIVES: Tissue engineering may provide a treatment for severe vocal fold scars. This study quantifies mechanical properties and demonstrates vibration of a tissue-engineered vocal fold cover replacement. METHODS: Tissue-engineered constructs were produced from fibrin and adipose-derived stem cells. Optimized bilayered constructs contained epithelial and mesenchymal cell phenotypes in a stratified geometry. For comparison, homogeneous constructs did not have epithelial differentiation. Elastic modulus was determined using indentation. Immunohistochemical labeling for type I collagen was performed. A bilayered construct was also tested in phonation in an excised larynx model. RESULTS: Bilayered vocal fold cover replacements had indentation moduli similar to human vocal fold covers (mean construct modulus 6.8 kPa). Collagen deposition occurred in the middle of the construct. Homogeneous constructs had a mean modulus of 8.3 kPa, and collagen was concentrated at the surface. An excised larynx with unilateral vocal fold cover replacement phonated and exhibited mucosal waves at physiologic airflow. CONCLUSION: Bilayered tissue-engineered constructs were produced that exhibited indentation modulus, microstructure, and vibration similar to that exhibited by human vocal fold covers. © 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. All rights reserved. T issue engineering may provide treatment for vocal fold scarring. We propose that replacing the entire vocal fold cover may be more effective than addressing only the lamina propria in severe cases. We have produced a stratified tissue-engineered construct resembling the vocal fold epithelium and lamina propria using adipose-derived stem cells (ASC) in fibrin. 1 With epidermal growth factor (EGF) and an air interface, the ASC differentiate into epithelial cells near the surface and mesenchymal cells within the construct bulk. We now assess whether this candidate cover replacement has mechanical and vibratory properties similar to the native vocal fold. Methods Institutional Review The UCLA Institutional Review Board approved the use of donated human lipoaspirate, cryoprecipitate, and cadaveric larynges. Fibrin-ASC Constructs ASC were isolated from lipoaspirate and cultured. 2 For fibrin constructs, cryoprecipitate was mixed with ASC and thrombin. 1 Three hundred L was polymerized within Transwell inserts (Cole-Parmer, Vernon Hills, IL), then concentrated ASC were added to the surface. Half of the constructs were cultured with an air interface and were supplied 10 ng/mL EGF and 10% fetal bovine serum in the culture medium (bilayered group). The remaining constructs had EGF-free culture medium and were submerged under liquid (homogeneous group). All were harvested at two weeks. For immunohistochemistry, samples were frozen, sectioned, and fixed onto slides. After blocking with goat serum, a rabbit antibody to type I collagen (DAKO, Denmark) was applied and detected with a goat-anti-rabbit fluorescein isothyocyanate-conjugated antibody. For a phonating construct, a 3 ϫ 1 cm rectangular well was scored in the base of a sterile culture dish. Cryoprecipitate-ASC-thrombin mixture was pipetted into the well. After the mixture gelled, additional concentrated ASC were added to the surface. Culture medium containing EGF bathed the gel on all sides but not on the surface. The sample was harvested at three weeks. Indentation A 1-mm indenter tip mounted onto a force transducer indented the construct surface in 0.025-mm steps until reach