Directly coaxial bioprinting of 3D vascularized tissue using novel bioink based on decellularized human amniotic membrane

Abstract

Despite several progressions in the biofabrication of large-scale engineered tissues, direct biopri nting of perfusable three-dimensional (3D) vasculature remained unaddressed. Developing a feasible method to generate cell-laden thick tissue with an effective vasculature network to deliver oxygen and nutrient is crucial for preventing the formation of necrotic spots and tissue death. In this study, we developed a novel technique to directly bioprint 3D cell-laden prevascularized construct. We developed a novel bioink by mixing decellularized human amniotic membrane (dHAM) and alginate (Alg) in various ratios. The bioink with encapsulated human vein endothelial cells (HUVECs) and a crosslinker, CaCl_2, were extruded via sheath and core nozzle respectively to directly bioprint a perfusable 3D vasculature construct. The various concentration of bioink was assessed from several aspects like biocompatibility, porosity, swelling, degradation, and mechanical characteristics, and accordingly, optimized concentration was selected (Alg 4 %w/v - dHAM 0.6 %w/v). Then, the crosslinked bioink without microchannel and the 3D bioprinted construct with various microchannel distances (0, 1.5 mm, 3 mm) were compared. The 3D bioprinted construct with a 1.5 mm microchannels distance demonstrated superiority owing to its 492 ± 18.8 % cell viability within 14 days, excellent tubulogenesis, remarkable expression of VEGFR-2 which play a crucial role in endothelial cell proliferation, migration, and more importantly angiogenesis, and neovascularization. This perfusable bioprinted construct also possess appropriate mechanical stability (32.35 ± 5 kPa Young's modulus) for soft tissue. Taking these advantages into the account, our new bioprinting method possesses a prominent potential for the fabrication of large-scale prevascularized tissue to serve for regenerative medicine applications like implantation, drug-screening platform, and the study of mutation disease.

Keywords: 3D-bioprinting; Alginate; Angiogenesis; Coaxial nozzle; Decellularized human amniotic membrane; HUVECs; Vascularization.