Development of biomaterials based on elastin like recombinamers for 3d bioprinting applications

Laburpena

The work presented in this doctoral thesis focuses on the development of unprecedented bioinks made from advanced biomaterials, such as Elastin Like Recombinamers (ELRs), for their use in 3D bioprinting. The innovative properties of the ELRs, that derive mainly from both their Inverse Transition Temperature and recombinant character, have enabled the creation of tailor made designs for the generation of highly mimetic tissue structures, providing innovative quality both in printing conditions and final pre-designed scaffolds. ELRs are proteinaceous biomaterials whose aminoacidic structure is inspired from the arrangement of the highly repetitive and well-conserved domains of the mammalian tropoelastin. They are produced via recombinant DNA technologies, which allow designing their sequences to show specific properties required for a certain application, through an extremely precise control over their amino acid composition. This means that this kind of biomaterials can be genetically customized for specific aimed applications. Moreover, the fact that ELRs are inspired in elastin, makes them acquire several of its interesting properties and, for this reason, ELRs have emerged as useful candidates for several biomedical applications showing an excellent biocompatibility, biodegradability, bioactivity and adjustable mechanical properties. Perhaps more interestingly for 3D bioprinting applications, is that they exhibit a smart behavior of thermo-responsiveness defined by the so-called Inverse Temperature Transition (ITT). So, in aqueous solution and below the transition temperature (Tt) of the recombinamer, the polymer chains remain soluble in a random coil conformation by hydrophobic hydration. If temperature rises above the Tt of the polymer, a hydrophobic folding is induced, leading to hydrogel formation when high concentration is used. This reversible phase transition can be seized for the ELRs deposition into 3D architectural matrices. Development of additive manufacturing technologies is currently revolutionizing the fields of tissue engineering and regenerative medicine, showing high future expectations. This technique has become one of the most innovative pioneer technologies in the search for novel biomimetic supports since it shows certain advantages with respect to its accuracy and repeatability. It also offers innovative possibilities for the construction of 3D architectural matrices not resolved up to now, such as the ability to design and introduce variable porosity in the constructions or to generate vasculature through the technique of printing with sacrificial inks. It also introduces other unprecedented architectural features, such as the possibility of inclusion of different materials and / or cell types within the same structure, as well as gradients, with a required positioning. All this novel structural features will enable a tailor-made tissue design. Complex parameters a biomaterial must comply with in order to be used for 3D bioprinting, refer both to its printing ability and to those characteristics that allow its use as a biomedical material. First of all, the biomaterial must show printability, influenced by its physicochemical parameters such as rheological properties (viscosity, pseudoplasticity, viscoelasticity and elastic limit) and cross-linking mechanisms. It must also be biocompatible and degradation kinetics must match the ability of the cells to form their own extracellular matrix and their degradation products must not be toxic. The structures formed by these materials must also show adequate structural and mechanical properties. All these strict requirements are not match in the already developed biomaterials for 3D bioprinting. So, structural and mechanical properties are the main disadvantages found in naturally derived materials, whereas synthetic materials which normally exhibit better printability, are unable to provide a proper cell proliferative environment. For this reason, this work arises with the desire to provide a progress into the bioink development paradigm by providing a useful and advanced biomaterial for the obtaining of highly developed 3D bioprinting scaffolds that show high shape fidelity without compromising cell behaviour. To date, there is no evidence of the use of ELRs as bioinks, used either as a main component or as a secondary component in the formation of multicomponent bioinks, which makes this research being innovative in the field of both 3D bioprinting and tissue engineering field. Considering the above, this research attempts to unveil the molecular mechanisms that will enable the ELRs to become printable as inks, focusing into the interplay between molecular forces allowing physical crosslinking of ELRs chains. In particular, the first chapter offers insight into the requirements that an ELR needs to demonstrate to be injectable within a bioprinting system, with a subsequent maintenance of the deposited shape. From the wide range of existing ELRs that can be potentially developed as new inks, this chapter is focused in those previously reported to exhibit a physical self-assembly hydrogel formation with structural stability among time. An intensive research over the formation of these ELR physical hydrogels related to their demonstrated printability allowed the determination of a favourable ELR formulation: a mixture between amphiphilic ELRs, with silk and leucine zipper domains, respectively, that provided a functional injectable hydrogel with advanced mechanical properties allowing the formation of 3D complex scaffolds via 3D printing. Printed scaffolds were also tested regarding their viability and cytocompatibility. The second chapter goes a step further by the designing of a new ink formulation based into the results obtained along the first chapter, where all the molecular domains where rationally introduced within the same ELR sequence, thus providing intramolecular interactions. In this way, the novel designed ELR ink formulation showed adequate properties for 3D printing, even when cellular components were added within. Several steps were used for the obtaining of the ELR ink, such as gene design, bioproduction into genetically modified microorganisms, protein purification, and elucidation of the physicochemical properties. Also, the novel ELR was tested for printability, scaffold formation and mechanical properties of the printed scaffolds. Finally, the ELR was used as a bioink mixed with cells where its biocompatible nature provided a protective environment throughout the bioprinting process, and where the viability, cytocompatibility and in vitro performance were tested. In this study, for the first time, the potential use of the ELRs as biomaterials for the construction of resolute cell-laden tissue matrices by additive manufacturing is demonstrated, being these biomaterials able to provide an extracellular matrix-like environment sufficient to induce cell proliferation and differentiation. The obtained results also highlight the applicability and novelty of the bioprinting of biomimetic ELR-based structures for advanced applications. In addition to their proven structural and biological properties, the synthetic production by genetic engineering of the ZS-EI-ELR recombinamer favors batch-to-batch homogeneity, thus removing one of the major drawbacks of natural bioinks. Moreover, this will also enable future bioink tunability given the ability to incorporate sequences of interest within the amino acid chain that may lead to innovative properties and bioactivities, thereby allowing the creation of tissue tailored bioinks.