"Tracheal damage is associated with the narrowing, weakening and discontinuity of the conductive part of the lower respiratory tract. Extensive defects cannot undergo end-to-end anastomosis and current approaches present poor outcomes due to weak mechanical properties, poor re-epithelialisation and vascularisation of the implanted graft. Herein, we investigated the use of collagen-based tubular scaffolds reinforced with 3D-printed synthetic polymer architectures for tracheal repair. A collagen and hyaluronic acid film covered the inner lumen (IL) of the scaffold, using CHyA-B scaffolds to support the formation of a respiratory epithelium. The 3D printed reinforced collagen porous outer layer (OL) of the scaffold was designed to support the growth of underlying tissues including cartilage and connective tissues as well as the formation of a vasculature network around the graft. The mechanical strength and ultrastructure of the tubular scaffolds was characterised and an approach for cellular seeding of the different layers of the scaffold was developed.
Tracheal scaffolds showed a 10-20 compressive MPa Young modulus with no significant decrease in mechanical strength following cyclic loading used to mimic respiratory patterns. Scaffold characterisation revealed a porous microarchitecture using scanning electron microscopy imaging with mean pore size of 169.7±11.2 µm suitable for cellular proliferation estimated via toluidine staining. A seeding process with the support of a custom-made device and 3D printed accessory parts was developed to achieve targeted epithelial seeding (Calu-3 bronchoepithelial cells) on the IL while the outer layer OL of the scaffold was populated with Wi38 lung-derived fibroblasts using different seeding densities under rotation. The growth of Wi38 cells on the OL was monitored for 7 days, showing successful cellular growth on the OL with no cellular attachment and growth in the IL using 6x105cells/cm2. Calu-3 cells were grown on the tubular scaffolds for 10 days, showing optimal cellular growth on the IL of the scaffold using 1.25x105 cells/cm2 with little attachment and growth of Calu-3 cells in the porous OL. Immunofluorescence imaging and quantification of the film from the IL further demonstrated cellular growth on the film with an estimated epithelial coverage of the film >60%.
Reinforcement of CHyA-B scaffolds with 3D-printed polymer architectures represents a suitable approach for the development of tissue-engineered tracheal grafts, showing adequate mechanical properties and an optimal porous structure to support the formation and growth of tracheal tissues. Moreover, a seeding procedure using a custom-made device was developed allowing successful cellular attachment and growth in the different layers of the tubular scaffold. The establishment of this targeted cellular seeding procedure holds potential to enable the clinical translation of tissue engineered tracheal grafts by facilitating differentiated pre-seeding strategies prior to implantation."