Development of magnetically-patterned anisotropic hydrogels for Xolography-based volumetric 3D printing to guide myocytes organization

Skeletal muscle (SkM) is a highly organized tissue displaying multiple levels of structural organization, whose development and contractile function is intimately related to its anisotropic structure. To engineer SkM in vitro models, scaffolds must mimic the native extracellular matrix (ECM) architecture, providing not only bio-chemical but also physical cues to promote myoblasts alignment and maturation. Hydrogels provide a water-rich, biomimetic 3D environment, and represent the most promising candidate materials to engineer scaffold for tissue engineering applications. However, the intrinsic isotropy inherent to their network is not capable of guiding cell alignment, limiting their applicability to those tissues characterized by high level of structural anisotropy, like skeletal muscle. One of the latest strategies explored to produce anisotropic hydrogel scaffolds is based on the incorporation and remote manipulation of magnetic nanoparticles (MNPs) inside the hydrogel liquid precursor. Under a uniform magnetic field, they spontaneously arrange into linear, chain-like aggregates aligned along the field lines, which are finally stabilized upon ‘sol-to-gel’ transition of the host matrix. These anisotropic structures can act as topographical cues to guide cells alignment, as largely demonstrated in the literature. Super paramagnetic iron oxide particles (SPIOPs) are the most used type of MNPs due to their high cytocompatibility. Beyond structuration at the microscale, hydrogels can be fabricated into the required shape by a wide range of additive manufacturing (AM) technologies, including direct ink writing (bioplotting) and vat photopolymerization. The use of AM strategies has rapidly spread in the field of tissue engineering to address the growing need to improve the control over the structural and mechanical properties of scaffolds at the macroscale, key factors in regulating cells behavior. The integration of AM technologies with the self-assembly approach will pave the way for the fabrication of constructs with structural control at multiple scale lengths. Within this framework, the aim of the work is to investigate a further step ahead in the development of advanced hydrogels, studying a magnetically patterned, cell-laden, hydrogel-based scaffold which can be processed via the innovative technology of Xolographic volumetric printing. The intended goal is to provide the hydrogel-photoresin with an inner microstrucure composed of anisotropic fibril arrangements of self-assembled SPIONs while preserving its printability. The research activities were conducted both at Politecnico di Torino (MPMNT research group) and at TUE in Eindhoven (the Netherlands), in the Dias Castilho’s group. In this Thesis work, first the principles of skeletal muscle engineering will be introduced (chapter 1), followed by a description of magnetic hydrogels (chapter 2) and additive manufacturing (chapter 3). Then, the experimental part will be showed, first explaining materials and methods (chapter 4) applied in this work and then reporting experimental results (chapter 5). Finally, a conclusion chapter will summarize the results obtained, discussing future developments.