Development of a tissue construct with spatially controllable stiffnessviaa one-step 3D bioprinting and dual-crosslinking process

3D bioprinting is a promising technology for manufacturing cellular constructs in which the complex characteristics of real tissues are faithfully represented under in vitro culturing conditions. Many efforts have been made to reproduce the numerous physical, chemical, and mechanical properties that are crucial determinants of cellular biological functions and behavior. Abrupt stiffness changes have been described in tumors, and higher stiffness has been reported for tumor regions within the same tissue. In this study, we developed a green synthetic route for alginate methacrylation with a degree of substitution higher than 60%, which was used for bioink formulations using HeLa cells as a proof of principle cell model. Alginate methacrylate (AlgMa) was characterized by H-1-NMR, FT-IR and MALDI TOF-TOF MS. A dual-step crosslinking procedure was developed to obtain bioprinted constructs with good cellular viability and tunable mechanical properties. A critical and systematic study of the effects of the photoinitiator concentration, photo-crosslinking process, and mechanical properties of the AlgMa-based construct on cell viability was conducted. Extensive rheological characterization of bioink formulations and bioprinted samples that differ in dual-step crosslinking procedures enabled the production of samples characterized by differential and spatially resolved mechanical properties within the same 3D bioprinted construct, approaching the heterogeneous stiffness observed in real tissues.