Engineering bioinks for 3D bioprinting of human tissue models as alternatives to in vivo testing

Three-dimensional (3D) bioprinting has emerged as a promising technique for the fabrication of tissue models, through the co-deposition of cells and biomaterials (bioinks). In line with the 3Rs principle, this approach enables the development of in vitro human tissue models as alternatives to in vivo testing. The goal of this PhD thesis was to optimize bioinks with tunable mechanical and biological properties, closely replicating the extracellular matrix of tissues. Interpenetrating polymer network (IPNs) hydrogels were engineered by combining two biopolymer networks such as alginate, gelatin methacrylate (GelMa), alginate methacrylate (AlgMa), and collagen. Additionally, a novel polymerization method, Frontal Polymerization (FP), was applied to synthesize semi-IPN hydrogels with tunable properties. To better understand which formulations performed best, various parameters were considered, such as (i) hydrogel concentration (ii) photoinitiator concentration, (iii) intensity and exposure time of UV light for the crosslinking. A deep rheological characterization was conducted on the bioink formulations to establish a correlation between mechanical properties and printability. According to the steps of 3D extrusion bioprinting, a first test to assess whether bioinks exhibit yield stress was performed by subjecting the materials to increasing shear stresses. Shear thinning properties were evaluated by measuring viscosity as a function of shear rate. Recovery tests were performed to assess the material’s ability to restore their elastic properties after deformation. Printing tests were conducted to assess the uniformity ratio and printability of different inks. The optimized bioinks enabled the fabrication of various tissue-specific 3D bioprinted models. These included a GelMA–alginate IPN-based liver model for hepatotoxicity screening, a GelMA–collagen IPN gut model allowing the incorporation of intestinal microbiota, and alginate composite hydrogels doped with bioactive glasses for bone tissue engineering. Overall, this work demonstrates that rheology-driven bioink optimization enable the fabrication of stable, printable, and biologically functional 3D bioprinted constructs.