3D bioprinting of pathophysiologically relevant in vitro models

In the field of Tissue Engineering (TE), Additive Manufacturing (AM), based on the deposition of material in a layer-by-layer fashion, represents a pivotal technology that allows for the fabrication of three-dimensional (3D) scaffolds with sophisticated designs. Among the diverse available technologies, microextrusion bioprinting is considered as part of the direct-ink-writing processes, relying on the tuning of the rheological properties of the ink, typically composed of hydrogel biomaterials mixed with living cells. Such printing process expects the bioink to be extruded directly in a gel-state without the addition of supporting structures that ensure the printability of the construct or microfluidic tools that induce crosslinking of the material during the printing step. In this context, the imperative challenge is the formulation of suitable inks with desirable features of both extrudability and post-printing stability. As a consequence, considerable combinations of biomaterials with complementary properties have been explored with the aim to achieve proper chemical, mechanical and biological features for the target tissue application. Multicomponent inks have therefore emerged as an optimal solution to overcome the drawbacks of single-hydrogel compositions, such as the limited printability of natural polymers or the lack of cell-specific activity of synthetic ones, thus integrating the advantages of each component and widening the biofabrication window. When distinct hydrogels are mixed together, the resulting polymeric blend can benefit from different crosslinking mechanisms, both chemical and physical, with the possibility to obtain a robust stabilization of the gel. Indeed, the single components of multicomponent bioinks can complement one another, account for what the other bioink material might be lacking, and act as a supplementing element that can assist in the formation of more complex tissue constructs. Several research groups have exploited this potential, managing to bioprint materials with enhanced printability and to fabricate functional human tissues with complex architectures. Exploiting the multicomponent approach, the present work reports two diverse ink formulations for the fabrication of liver and skin tissue models by means of a microextrusion bioprinting technique. Both bioinks are Pluronic F127-based hydrogels, with the aim to take advantage from its thermosensitive and rheological properties, acting as a template agent and promoting the extrusion process of low-viscosity polymers such as alginate (liver model) and gelatin methacrylate (skin model). Indeed, Pluronic/alginate thermogel was adopted for the fabrication of a 3D hepatic construct embedded with HepG2 cells with the aim to investigate liver-specific metabolic activity and perform a hepatotoxicity testing using acetaminophen as model drug. Results demonstrated the potential of the present in vitro hepatic model as an innovative platform for drug screening, compared to traditional bidimensional culture systems. Pluronic/gelatin methacrylate hydrogel was instead used to bioprint skin constructs with embedded human fibroblasts as a support for keratinocytes adhesion and growth. The biofabricated proof-of-principle model proved to be suitable for enduring a long-term culture of skin cells, paving the way for the development of alternative platforms to animal experimentation for drug and cosmetic testing in full agreement with the principles of the "three Rs" (Replacement, Reduction and Refinement). The work is divided into two different parts: part I consists of three chapters and it focuses on the current state of the art on Additive Manufacturing of tissue models, particularly on liver and skin tissue. Part II is devoted to the research activities, presenting the experimental methodology conducted for the biofabrication of hepatic and cutaneous in vitro models, and reporting the main results achieved within the project. Finally, the last chapter sums up and collects all the conclusions and proposes future developments to the present work.