Dual crosslinkable bioink for direct and embedded 3D bioprinting at physiological temperature

Abstract

Bioprinting at physiological temperature (PT) is desirable to maintain cell viability during and after printing, especially for tissue and organ scale constructs requiring a long fabrication time. Typically, bioinks show a temperature viscosity dependence and exhibit poor printability at PT, limiting construct complexity and shape fidelity. Embedded bioprinting offers opportunities to print low viscosity bioinks, however, the consideration of PT is often neglected. In this study, a dual thermoresponsive and photocrosslinkable bioink was developed comprising gelatin methacryloyl (GelMA) and either methylcellulose (MC) or methylcellulose methacrylate (MCMA). This bioink serves as both a direct extrusion bioink and as a support bath for embedded bioprinting. Both MC and gelatin show synergistic thermosensitive rheological properties which was exploited to enable bioprinting at both room temperature and PT to create a semi-interpenetrating or interconnected polymer network with tuneable properties. The bioinks show sol–gel transitions at temperatures of < 27 °C and > 33 °C, representing the contribution from GelMA and MC/MCMA, respectively, and improved thixotropic, and self-healing behaviour at PT. These rheological properties significantly improve printability at a range of temperatures (18–37 °C) and allows the bioink to function as a support bath at PT. Moreover, higher cell viability (>90 %) post-bioprinting was observed in PT bioprinted constructs. The cell response in bioprinted constructs was dependent on bioink composition and cell density, with low polymer concentration and increased cell densities favouring the spreading and proliferation of adipose-derived stem cells. Acting as a support bath, the bioink enabled fabrication at PT of complex cell-laden structures through embedded bioprinting. This allowed spatial patterning of a variety of cell types and perfusable channels with the support bath acting as a matrix to provide long-term support and as a reservoir of cells. The bioinks successfully expand bioprinting capability at PT for both direct and embedded bioprinting and has promising potential to fabricate large-scale tissue models for tissue engineering applications.