Development of starch-based support material alternately extruded with gelatin-based bioinks for 3D bioprinting application

The use of support materials is crucial for the 3D bioprinting of low-viscosity bioinks, which yield soft hydrogel constructs susceptible to deformation under their weight. In this study, we developed a starch-based support material that provides structural support during printing and supplies hydrogen peroxide (H₂O₂), for printing cell-laden constructs from low-viscosity bioinks (4.4–53.1 mPa s at 1 s⁻¹ shear rate) composed of a gelatin derivative possessing phenolic hydroxyl moieties (gelatin-Ph), horseradish peroxidase (HRP), and cells. Importantly, the support material can be selectively and gently removed using α-amylase, a biocompatible enzyme, without harming the construct or encapsulated cells, which is a significant advancement over conventional methods of removing support systems. 3D constructs were fabricated by alternately extruding bioinks containing 5.0 w/v% gelatin-Ph and 10 U/mL HRP with a support material consisting of 16.7 w/w% starch and 10 mM H₂O₂. Immortalized human bone marrow-derived mesenchymal stem cells encapsulated within the constructs showed >80 % viability after printing and exhibited an elongated morphology and proliferation, while maintaining their stemness over 14 days of culture. The cells underwent osteogenic differentiation when cultured in a differentiation medium, as evidenced by the calcium deposition, alkaline phosphatase activity, and expression of osteogenic genes, demonstrating the potential of the proposed approach for tissue-engineering applications.