Multi-tissue type condensations for trachea tissue regeneration via individual cell bioprinting

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Abstract As a result of prolonged intubation, tracheostomy, external trauma, penetrating fragment projectiles, gunshot wounds and improvised explosive devices during combat, and benign or malignant tumors, many soldiers and veterans in the US military suffer from severe trachea stenosis or damage that can cause complete airway failure, Since there is no successful long-term treatment for long-segment tracheal stenosis or damage, tissue engineering strategies have been explored to develop neotracheas using different combinations of biomaterials and cell sources. However, biomaterial scaffold-based approaches often interfere with critical cell-cell interactions, cell proliferation and new extracellular matrix production that are important during the formation of functional trachea tissue. A functional replacement trachea must retain (1) radial rigidity to prevent restenosis, (2) anastomose with host vasculature to adequately provide nutrients to the implant, and (3) contain respiratory epithelium to provide a protective mucosal layer. Combining three-dimensional (3D) bioprinting technologies with scaffold-free tissue engineering principles presents a powerful platform for engineering a multi-tissue functional trachea, and would circumvent the aforementioned limitations of scaffold-based approaches. This proposal aims to leverage the benefits of our recently developed individual cell-only 3D bioprinting technology, which allows for printing of complex and high-resolution cell condensation-based tissue constructs to engineer functional tracheas. We plan to print scaffold-free, multi-tissue neotracheas using multiple discrete individual cell-only bioinks for spatially distinct differentiation of tissue types driven by spatially controlled presentation of tissue- specific growth factors. Construct self-assembly will be driven by the condensation of autologously sourced human mesenchymal stem cells (hMSCs) for cartilaginous tissue and autologous endothelial progenitor cells and hMSCs for prevascular tissue, with autologous human bronchial epithelial cells applied to line the lumen of the neotrachea. Specifically, this proposal aims to (1) examine the role of the physical properties of the microgel support slurry on cell-only bioink printing, condensation formation/maintenance, and chondrogenesis of the 3D bioprinted structures, (2) 3D bioprint cartilage ring constructs with chondrogenic bioink and prevascularized ring constructs with vasculogenic bioink using the individual cell-only bioprinting technology, and (3) engineer prevascularized and epithelized tracheal tissue with chondrogenic and vasculogenic bioinks using the individual cell-only bioprinting technology. As an exploratory aim, the capacity of the engineered tracheas to restore airway functionality will be evaluated in an animal defect model. This work ultimately seeks to utilize a facile and flexible individual cell-only bioink 3D printing platform to engineer a patient-specific replacement trachea that provides requisite physiologic and mechanical properties for replacement in those that are affected by long-segment tracheal stenosis. The inherent flexibility of this individual cell-only 3D printing platform to create complex structures composed of multiple spatially distinct tissue types can be leveraged to develop other multi-tissue organs.