Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.
Tracheal reconstruction is indicated in cases of malignancy, traumatic injury, and subglottic or tracheal stenosis. Recent progress in airway transplantation has provided renewed optimism for potential solutions for defects involving more than half of the tracheal length in adults or one-third of the tracheal length in children. Biologic scaffolds derived from decellularized tissues and organs have shown great promise in tracheal allotransplantation, and cyclical decellularization techniques have been hypothesized as abrogating the need for immunosuppressive therapy. In this study, we performed a direct comparison of three decellularization protocols (Protocols A, B, and C) previously described in the literature, two of which were described in tracheal tissue (Protocols A and B). We concentrated on the immunogenicity within the epithelium and mucosa, quantified and qualified the extracellular matrix (ECM) components, and performed compliance measurements on large circumferential decellularized tracheal scaffolds following cyclical decellularization techniques using all three protocols. Quantitative measurements of glycosaminoglycans (GAGs) showed a significant decrease in the mucosal component following 17 cycles of all 3 protocols as well as a significant decrease of GAGs in the cartilaginous component following cycles 1, 9, and 17 of Protocol A and cycle 17 of Protocol C. Compliance measurements were also shown to be different between the protocols, with grafts becoming more compliant at physiologic pressures after cyclical decellularization with Protocols A and B and slightly less compliant but remaining similar to native trachea using Protocol C. Positive staining for anti-major histocompatibility complex Class I (anti-MHCI) and anti-MHCII remained within the submucosal glandular components despite multiple cycles of decellularization using all three protocols. This study illustrated that there are significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the decellularization protocol. Protocol B was shown to maintain the GAGs components despite an increase in tracheal compliance, while Protocol C decreases GAGs components following multiple cycles, despite showing a tracheal compliance resembling that of the native trachea at physiologic airway pressures.
Summary:A recent revival of global interest for reconstruction of long-segment tracheal defects, which represents one of the most interesting and complex problems in head and neck and thoracic reconstructive surgery, has been witnessed. The trachea functions as a conduit for air, and its subunits including the epithelial layer, hyaline cartilage, and segmental blood supply make it particularly challenging to reconstruct. A myriad of attempts at replacing the trachea have been described. These along with the anatomy, indications, and approaches including microsurgical tracheal reconstruction will be reviewed. Novel techniques such as tissue-engineering approaches will also be discussed. Multiple attempts at replacing the trachea with synthetic scaffolds have been met with failure. The main lesson learned from such failures is that the trachea must not be treated as a “simple tube.” Understanding the anatomy, developmental biology, physiology, and diseases affecting the trachea are required for solving this problem.
Tracheal transplantation with a long-segment recellularized tracheal allograft has previously been performed without the need for immunosuppressive therapy. Recipients' mesenchymal stromal cells (MSC) and tracheal epithelial cells (TEC) were harvested, cultured, expanded, and seeded on a donor trachea within a bioreactor. Prior techniques used for cellular seeding have involved only static-seeding methods. Here, we describe a novel bioreactor for recellularization of long-segment tracheae. Tracheae were recellularized with epithelial cells on the luminal surface and bone marrow-derived MSC on the external surface. We used dynamic perfusion seeding for both cell types and demonstrate an increase in both cellular counts and homogeneity scores compared with traditional methods. Despite these improvements, orthotopic transplantation of these scaffolds revealed no labeled cells at postoperative day 3 and lack of re-epithelialization within the first 2 weeks. The animals in this study had postoperative respiratory distress and tracheal collapse that was incompatible with life.
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