Periodic Reporting for period 3 - SupraCTRL (From mechanical control to shape-shifting in supramolecular biomaterials to guide stem cell fate)
Periodo di rendicontazione: 2023-09-01 al 2025-02-28
The biochemical and biophysical cues of the stem cell environment that act in a concerted and spatiotemporal manner lead to the formation of the 200 cell types and organs of the human body, but how this precisely occurs remains unclear and it is necessary to guide their production for use in the biomedical area. Standard differentiation protocols in vitro mimic known stages in development by the timed addition of biochemical cues on 2D substrates, however these protocols lack the complexity of the 3D natural extracellular matrix (ECM), with its mechanical character that evolves in time. Supramolecular materials can recapitulate the structural and dynamic character of the ECM being based on non-covalent interactions. Their mechanical soft character can mimic embryonic microenvironment for induced pluripotent stem cell (iPSC) culture but renders them unable to mimic stiff and tough tissues.
Double networks using covalent polymers have demonstrated to achieve such mechanical properties, however these materials lack the cytocompatibility for use in 3D cell culture. The ERC project will address these challenges through objectives that include the synthesis of hybrid covalent-supramolecular polymer networks using biocompatible chemical and light-activated ligation approaches and the application of the materials to guide the fate of iPSCs to cardiomyocytes by controlling their mechanical properties in time. Subsequently, the cell laden materials will be interfaced with biomechanical devices to exploit their unique mechanical properties, and the materials will also be 3D-bioprinted with cells to prepare an actuatable culture platform in the form of a miniature beating heart ventricle. These advanced culture platforms based on hybrid-covalent supramolecular materials that go from soft to stiff and tough in time and space with shifting-shapes, with the potential to decouple the presentation of bioactive cues in an integrated manner, will provide uncharted opportunities to understand the spatiotemporal evolution of active and passive mechanical cues in development from cell to organ. The developed strategies within this ERC project will be essential for the development of cardiac models at various length scales with improved accuracy for in vitro drug screening, toxicity testing and disease modeling that will have societal impact on healthcare.
Double networks using covalent polymers have demonstrated to achieve such mechanical properties, however these materials lack the cytocompatibility for use in 3D cell culture. The ERC project will address these challenges through objectives that include the synthesis of hybrid covalent-supramolecular polymer networks using biocompatible chemical and light-activated ligation approaches and the application of the materials to guide the fate of iPSCs to cardiomyocytes by controlling their mechanical properties in time. Subsequently, the cell laden materials will be interfaced with biomechanical devices to exploit their unique mechanical properties, and the materials will also be 3D-bioprinted with cells to prepare an actuatable culture platform in the form of a miniature beating heart ventricle. These advanced culture platforms based on hybrid-covalent supramolecular materials that go from soft to stiff and tough in time and space with shifting-shapes, with the potential to decouple the presentation of bioactive cues in an integrated manner, will provide uncharted opportunities to understand the spatiotemporal evolution of active and passive mechanical cues in development from cell to organ. The developed strategies within this ERC project will be essential for the development of cardiac models at various length scales with improved accuracy for in vitro drug screening, toxicity testing and disease modeling that will have societal impact on healthcare.
The major achievements within the project so far involve the introduction of bioactivity into the squaramide hydrogels through supramolecular monomers that permit bioconjugation with peptides. We have shown that cell differentiation can occur within the peptide-functionalized materials using HepG2 spheroids. Using our earlier expertise in working with dithiolanes on covalent polymers and their crosslinking through light activation for 3D cell culture, we then applied this unit in supramolecular materials in the ERC project. We demonstrated that supramolecular monomers with dithiolane units can be introduced into these materials to prepare hydrogels that can be used for 3D cell culture. The ring strained cyclic disulfides can be addressed with light enabling spatial and temporal patterning of mechanics and bioactivity within the materials. These changes in network properties are recognized by the cells within the materials triggering changes in their morphology and migration. We have also further examined the potential to prepare disulfide-cross linked networks in the absence of light using oxidized dithiolanes or cyclic thiosulfinates on covalent polymers. Through mixing with a macromonomer containing a free thiol, dynamic covalent networks with tunable viscoelasticity can be engineered for the culture of sensitive or non-proliferative cell types such as induced pluripotent stem cell cardiomyocytes. The dynamic character of the materials permits the cells to show native cardiomyocyte behaviours such as beating, elongation and alignment. The development of these various chemical strategies enables us to prepare more complex hybrid networks based on the materials.
The current achievements in the project advance the field of synthetic polymers used as biomaterials demonstrating the potential of cyclic disulfides and thiosulfinates as crosslinking chemistries. Cells recognize these complex mechanical and bioactive changes within the materials when these latent units are triggered resulting in specific behaviours. Importantly, the introduced chemical handles permit the modification of the materials in space and time that are essential to mimic processes that occur in development and disease. The results obtained for the single networks thus far will be essential for their mixing as hybrids and sets the stage for their further biofabrication with cells towards the end of the project.