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

• Applicants: Professor Dr.-Ing. Horst Fischer; Dr. Juliane Schuphan
• Subject Area: Biomaterials
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 403762164

Achieving precise replication of natural tissue structures is essential in the development of biomaterials for bone tissue engineering. Nanofibers made of various biopolymers have been shown to improve cell adhesion, proliferation, and differentiation. Specifically, rod-shaped plant viruses nanoparticles (VNPs) have proven to be excellent candidates for nanocomposites in hydrogels, because of their high aspect ratio. In the first phase of the project, Potato virus X (PVX) was genetically modified to present mineralization- and osteogenesis-inducing peptides, which mimic non-collagenous proteins. This modification resulted in improved biomineralization and osteogenesis of human mesenchymal stem cells (hMSCs) in both 2D and 3D environment. In the second phase of the project, it was demonstrated that the combination of mineralization-VNPs and VNPs presenting RGD can achieve a synergistic osteogenic effect without increasing VNP concentrations. Additionally, we developed several PVX nanoparticles that were genetically modified to present angiogenic peptides or peptides that have both angiogenic and osteogenic effects at a concentration of only 1 ng/ml. Considering the advantages that engineered plant VNPs offer, they could be combined with plug-and-display technologies to easily incorporate multiple osteogenic and angiogenic peptides into a VNP-based network without the need for chemical crosslinkers or physical stimulation such as heat and UV exposure. Furthermore, the use of VNP networks obtained through such technologies has the potential to produce hydrogels themselves consisting of a network of synergistically acting functional peptides from the first two phases of the project. In the third and final phase, we therefore intend to co-culture hMSCs and human umbilical vein endothelial cells (HUVECs) within the VNP network-based hydrogels. Interactions of biomimetic peptides (referred to as connectors) within the VNP network and corresponding cell receptors will be investigated using confocal imaging, immunoprecipitation, and western blotting techniques. The osteogenic and angiogenic response of the cells will be explored using ELISA-based assays for supernatant analysis, and qPCR, and western blotting for cell lysate analysis. The viscosity and stiffness of the VNP-network hydrogels will be determined for various concentrations of VNPs and connectors. These factors are crucial in influencing cell behavior. Finally, the suitability of the VNP network for 3D bioprinting will be investigated using three different bioprinting methods, i. e. microextrusion, inkjet, and acoustic bioprinting.

• DFG Programme: Research Grants