Biomineralizing three-dimensional bioprinted hydrogels for bone tissue replacement using dedicated peptides in high local concentrations presented on biotechnologically modified plant virus nanoparticles (PlantVirusBone)

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A suitable biomaterial for tissue engineering must meet certain challenges such as biodegradability, cytocompatibility and bioactivity, but most hydrogels lack crucial biochemical cues for cell interaction. Current research therefore focuses on novel hydrogel blends which resemble the structure of the extracellular matrix and allow biochemical attachment of the encapsulated cells. In the first project phase, Potato virus X (PVX) was engineered to present mineralization- and osteogenesis-inducing peptides (MIPs), mimicking non-collagenous proteins (NCPs), which improved the human mesenchymal stem cells (hMSCs) osteogenesis and biomineralization in both 2D and 3D environments. Plant virus nanoparticles (VNPs) displaying different coverages of MIPs, demonstrated that the mineralization effect and cell interaction is the result of the high local density of presented MIPs, highlighting VNPs as interesting platforms for biochemical cues. Moreover, they showed a good cytocompatibility and a high retention inside the hydrogels.Another requirement for a biofunctional tissue replacement is a sufficient vascularization. Thus, the proposed second project phase aims to develop a regenerative bone tissue with multiple physicochemical properties to simultaneously provide oxygen and nutrient supply and promote osteogenesis. A synergistic osteogenic and vasculogenic effect in both 2D cell culture and 3D cell-embedded hydrogels can be achieved by employing different VNP modifications with various peptides derived from NCPs and vascular endothelial growth factor. VNPs will be modified with novel strategies, including different ribosomal skipping sequences, and covalent plug-and-display systems. The latter enables VNPs to interconnect to large bundles, which can produce a hydrogel itself consisting of a network of synergistically acting functional peptides. A capillary-like network will be induced by co-culturing hMSCs and human umbilical vein endothelial cells on 2D VNP-coated surfaces as well as in VNP-laden 3D hydrogels. Osteogenic and angiogenic capacities of the cells will be evaluated especially by real-time PCR, migration assay and fluorescence imaging. The mechanical properties, vital factors for influencing the cell behavior, will be tuned by changing the physical or chemical composition of hydrogels. Viscosity and stiffness of the VNP-laden hydrogels will be determined for different VNPs and hydrogels. Finally, bioprinting technology will be applied to generate a tissue substitute with spatially defined VNPs, cells and materials organization. The influence on the cell response will be studied in detail.