Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts.

中文翻译：

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从原代人细胞和脱细胞的喉支架创建喉移植物。

当前的喉气管节段的重建方法不能代替人喉的复杂功能。重建的生物工程方法受到喉部复杂的组织分隔的限制。我们试图通过在统一的培养基条件下对人原代细胞进行脱细胞的脱细胞犬喉支架进行生物工程喉移植来克服这一限制。首先，我们开发了经过9天的去污剂灌注脱细胞作用产生的喉咙支架，并保留了它们的糖胺聚糖含量和天然喉的生物力学特性。在大鼠中皮下植入14天后，支架没有引起CD3淋巴细胞反应。然后，我们开发了一种均匀的培养基，该培养基在初始生长阶段后的5天内会增强内皮屏障。同时，这种培养基支持气道上皮细胞和骨骼成肌细胞的生长，同时保持它们的完全分化和成熟潜能。然后，我们将均匀的培养基成分应用于从颈动脉和颈外静脉注入内皮细胞的整个喉架，并产生内皮化的动脉和静脉血管床。在相同的培养基上，我们将上皮单层生物工程改造到喉粘膜上并重新填充了固有的喉肌。然后，我们能够在免疫缺陷小鼠的肌内移植模型中证明早期肌肉形成。