Tissue-engineering the larynx: Effect of decellularization on human laryngeal framework and the cricoarytenoid joint

Decellularization approaches have been commonly used as alternative techniques to reconstruct tissues. However, due to the complex tissue compartmentation of the larynx, the decellularization process may not retain the characteristics necessary for the successful recreation of the larynx. The aim of this study was to assess the effect of the decellularization process on the framework of the human cadaveric larynx generally and the cricoarytenoid joint specifically. In this work, five freshly frozen human cadaveric larynges were decellularized utilizing a protocol that was previously demonstrated to be effective in decellularizing a porcine larynx. The decellularization protocol included: biological, chemical, and physical decellularization methods. Each specimen served as its own control to assess changes after decellularization. Studies and measurements included: histological, using Hematoxylin and Eosin (H&E) and Live/Dead™ stains; DNA quantification; micro-computed tomography (μ-CT) imaging; and biomechanical testing of the cricoarytenoid joints. The decellularization protocol took 12 days for each specimen. Microscopy of H&E stained samples demonstrated substantial removal of cells with preservation of the extracellular matrix that was more evident in cartilage than muscle specimens. Confocal microscope images of Live/Dead™ stained specimens also demonstrated almost complete removal of cells. Pre-decellularization cartilage-DNA quantity range was 27.0 to 336.8 ng/mg while post-decellularization DNA quantity range was 0 to 30.4 ng/mg (p = 0.031). For muscles, pre-decellularization DNA quantity range was 150.0 to 3,384.6 ng/mg, while post-decellularization DNA quantity range was 0 to 45.5 ng/mg (p = 0.031). μ-CT demonstrated preservation of the cartilaginous framework with a slight reduction of cricoarytenoid joint space. Furthermore, μ-CT demonstrated no significant reduction in the Housefield unit (p = 0.25) and mineral density (p = 0.25) after decellularization. Biomechanical testing demonstrated a non-significant reduction of forces required for anterior displacement of the arytenoid (mean reduction of forces, 0.1 ± 0.2 N, p = 0.16) and forces required for posterior displacement of the arytenoid (mean reduction of forces, 0.2 ± 0.3 N, p = 0.05). This study demonstrates effective decellularization of human larynges as evidenced by significant DNA depletion and preservation of extracellular matrix, which are outcomes that are required for a biological scaffold to regenerate a non-immunogenic larynx. The decellularization process caused minimal weakness in the cricoarytenoid joints due to treatment with multiple detergents and enzymes in the decellularization protocol.