Tissue-engineered larynx using perfusion-decellularized technique and mesenchymal stem cells in a rabbit model

Conclusion: Reseeding mesenchymal stem cells (MSCs) into the decellularized laryngeal muscle matrix for construction of a tissue-engineered larynx is feasible. This in vivo maturation in the omentum could be the first step before in situ implantation of the construct. This construct could facilitate a tissue-engineered method for laryngeal reconstruction. Objectives: The extracellular matrix (ECM) and MSCs have been widely used for organ reconstruction. Our study aimed to prepare a soft tissue decellularized laryngeal scaffold with intact laryngeal cartilages utilizing a perfusion-decellularized technique, reseeding cells on it, and then construct a recellularized larynx. Methods: Perfusion-decellularized larynges were obtained from 20 rabbits by perfusion of the common carotid arteries with detergents. Twelve perfused larynges were observed by macroscopic visualization, histological examination, scanning electron microscopy (SEM), and cartilage viability. The remaining eight perfusion constructs were reseeded with induced MSCs aspirated from eight receptor rabbits. Composites were transferred into greater omentums of receptor rabbits after adherence for 1 day in vitro. Rabbit larynges were harvested after 4 weeks and 8 weeks, respectively. Macroscopic visualization, histological examination, and immunohistochemistry were performed. Results: Larynges perfused by sodium dodecyl sulfate became transparent after 2 h of perfusion. Histology and SEM indicated that the perfusion method showed a better decullularized effect. Almost no intact cells or nuclei were found, while more pores and collagen fibers were retained in the decellularized matrix. The chondrocyte vitality assay indicated that chondrocyte vitality was high. Vascularization was clearly seen by 4 weeks and relatively integrated cartilage frameworks remained by 8 weeks. Histological and immunohistochemical examinations clearly showed muscle bundles and vessels.