Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.

Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.