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

一种适合组织工程的生物材料必须满足某些挑战，如生物降解性、细胞相容性和生物活性，但大多数水凝胶缺乏关键的细胞相互作用的生化信号。因此，目前的研究集中在新型水凝胶混合物上，这种水凝胶类似于细胞外基质的结构，并允许被包裹的细胞进行生化附着。在第一个项目阶段，马铃薯X病毒(PVX)被设计成呈现矿化和成骨诱导多肽(MIP)，模拟非胶原蛋白(NCP)，促进人骨髓间充质干细胞(HMSCs)在2D和3D环境中的成骨和生物矿化。展示不同覆盖度的MIP的植物病毒纳米粒(VNPs)表明，矿化效应和细胞相互作用是MIP局部密度高的结果，突出了VNPs作为有趣的生化线索平台的重要性。此外，它们表现出良好的细胞相容性和在水凝胶中的高保留率。生物功能组织替代的另一个要求是充分的血管形成。因此，拟议的第二阶段项目旨在开发一种具有多种物理化学性质的再生骨组织，以同时提供氧气和营养供应，并促进成骨。在2D细胞培养和3D细胞包埋水凝胶中，通过对来自NCPs和血管内皮生长因子的不同多肽进行不同的VNP修饰，可以获得协同的成骨和血管生成效应。VNPs将用新的策略进行修饰，包括不同的核糖体跳过序列和共价即插即用系统。后者使VNPs能够与大束相互连接，从而产生水凝胶本身，该水凝胶由协同作用的功能多肽网络组成。将人骨髓间充质干细胞与人脐静脉内皮细胞在2D VNP涂层表面以及负载VNP的3D水凝胶中共培养，可诱导形成毛细血管样网络。细胞的成骨和血管生成能力将通过实时定量聚合酶链式反应、迁移试验和荧光成像进行评估。机械性能是影响细胞行为的重要因素，将通过改变水凝胶的物理或化学组成来调整。对于不同的VNPs和水凝胶，将测定其粘度和硬度。最后，生物打印技术将被应用于产生具有空间定义的VNPs、细胞和材料组织的组织替代品。将详细研究这些因素对细胞响应的影响。

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.