Combined Use of Detergents and Ultrasonication for Generation of an Acellular Pig Larynx.

The larynx is a fairly complex organ comprised of different muscles, cartilages, mucosal membrane, and nerves. Larynx cancer is generally the most common type of head and neck cancer. Treatment options are limited in patients with total or partial laryngectomy. Tissue-engineered organs have shown to be a promising alternative treatment for patients with laryngectomy. In this report we present an alternative and simple procedure to construct a whole pig larynx scaffold consisting of complete acellular structures of integrated muscle and cartilage. Larynges were decellularized (DC) using perfusion-agitation with detergents coupled with ultrasonication. DC larynges were then characterized to investigate the extracellular matrix (ECM) proteins, residual DNA, angiogenic growth factors, and morphological and ultrastructural changes to ECM fibers. After 17 decellularization cycles, no cells were observed in all areas of the larynx as confirmed by hematoxylin and eosin and DAPI (4',6-diamidino-2-phenylindole) staining. However, DC structures of dense thyroid and cricoid cartilage showed remnants of cells. All structures of DC larynges (epiglottis [p < 0.0001], muscle [p < 0.0001], trachea [p = 0.0045], and esophagus [p = 0.0008]) showed DNA <50 ng/mg compared with native larynx. Immunohistochemistry, Masson's trichrome staining, and Luminex analyses showed preservation of important ECM proteins and angiogenic growth factors in DC larynges. Compared with other growth factors, mostly retained growth factors in DC epiglottis, thyroid muscle, and trachea include granulocyte colony-stimulating factor, Leptin, fibroblast growth factor-1, Follistatin, hepatocyte growth factor, and vascular endothelial growth factor-A. Scanning electron microscopy and transmission electron microscopy analysis confirmed the structural arrangements of ECM fibers in larynges to be well preserved after DC. Our findings suggest that larynges can be effectively DC using detergent ultrasonication. ECM proteins and angiogenic growth factors appear to be better preserved using this method when compared with the native structures of larynges. This alternative DC method could be helpful in building scaffolds from dense tissue structures such as cartilage, tendon, larynx, or trachea for future in vitro recellularization studies or in vivo implantation studies in the clinic.

Replacement of a tracheal stenosis with a tissue-engineered human trachea using autologous stem cells: a case report.

Cell-based therapies involving tissue engineering represent interesting and potentially important strategies for the treatment of patients with various disorders. In this study, using a detergent-enzymatic method, we prepared an intact three-dimensional scaffold of an extracellular matrix derived from a human cadaver donor trachea, which we repopulated with autologous stem cells and implanted into a 76-year-old patient with tracheal stenosis including the lower part of the larynx. Although the graft provided the patient with an open airway, a week after the surgery, the mucous membrane of the graft was covered by a 1-2 mm thick fungal infection, which was treated with local and systemic antifungal therapy. The airway lumen was postoperatively controlled by fiber endoscopy and found stable and sufficient. However, after 23 days, the patient died due to cardiac arrest but with a patent, open, and stable tracheal transplant and intact anastomoses. Histopathological results of the transplanted tracheal graft during autopsy showed a squamous but not ciliated epithelium, neovascularization, bundles of α-sma-positive muscle cells, serous glands, and nerve fibers with S-100-positive nerve cells in the submucosa and intact chondrocytes in the cartilage. Our findings suggest that although autologous stem cells-engineered tracheal matrices may represent a tool for clinical tracheal replacement, further preclinical studies are required for generating functional airway grafts and long-term effects of such grafts.