Department of Dental Materials and Biomaterials Research, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany.
Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
Acta Biomater. 2017 Oct 15;62:317-327. doi: 10.1016/j.actbio.2017.08.039. Epub 2017 Aug 30.
Bionanoparticles based on filamentous phages or flexuous viruses are interesting candidates for meeting the challenges of tailoring biomineralization in hydrogel-based bone tissue substitutes. We hypothesized that hydroxyapatite crystal nucleation and matrix mineralization can be significantly increased by mineralization-inducing (MIP) and integrin binding motif (RGD) peptides presented on biomimetic nanoparticles. In this study, Potato virus X (PVX), a flexible rod-shaped plant virus was genetically engineered to present these functional peptides on its particle surface. Recombinant PVX-MIP/RGD particles were isolated from infected Nicotiana benthamiana plants and characterized by western blot, SEM, TEM, and TPLSM in MSC cultures. The presence of RGD was proven by cell attachment, spreading, and vinculin cluster analysis, and MIP by in vitro mineralization and osteogenic differentiation assays. Thus the tailored surface of genetically engineered PVX forms fibril-like nanostructures which enables enhanced focal adhesion-dependent cell adhesion, and matrix mineralization verified by Alizarin. Hydroxyapatite crystal nucleation is supported on recombinant PVX particles leading to a biomimetic network and bundle-like structures similar to mineralized collagen fibrils. In conclusion, the recombinant flexuous PVX nanoparticles exhibit properties with great potential for bone tissue substitutes.
A suitable biomaterial for tissue engineering should be able to mimic the endogenous extracellular matrix by presenting biochemical and biophysical cues. Novel hydrogel-based materials seek to meet the criteria of cytocompatibility, biodegradability, printability, and crosslinkability under mild conditions. However, a majority of existing hydrogels lack cell-interactive motifs, which are crucial to modulate cellular responses. The incorporation of the plant virus PVX to the hydrogel could improve functions like integrin-binding and mineralization due to peptide-presentation on the particle surface. The tailored surface of genetically engineered PVX forms fibril-like nanostructures which enables enhanced focal adhesion-dependent cell adhesion and matrix mineralization and offers great potential for the development of new hydrogel compositions for bone tissue substitutes.
基于丝状噬菌体或柔韧病毒的纳米粒子是满足定制水凝胶基骨组织替代物中生物矿化挑战的有趣候选物。我们假设，通过在仿生纳米粒子上呈现矿化诱导（MIP）和整合素结合基序（RGD）肽，可以显著增加羟基磷灰石晶体成核和基质矿化。在这项研究中，马铃薯病毒 X（PVX），一种灵活的杆状植物病毒，经过基因工程改造，在其粒子表面呈现这些功能肽。从感染的 Nicotiana benthamiana 植物中分离出重组 PVX-MIP/RGD 颗粒，并通过 Western blot、SEM、TEM 和 TPLSM 在 MSC 培养物中进行表征。通过细胞附着、扩散和 vinculin 簇分析证明了 RGD 的存在，通过体外矿化和成骨分化试验证明了 MIP 的存在。因此，经过基因工程改造的 PVX 的定制表面形成了原纤维样纳米结构，从而增强了依赖于焦点附着的细胞附着，并通过茜素红验证了基质矿化。在重组 PVX 颗粒上支持羟基磷灰石晶体成核，导致形成仿生网络和束状结构，类似于矿化的胶原纤维。总之，重组柔韧 PVX 纳米粒子表现出具有巨大潜力的骨组织替代物的特性。
合适的组织工程材料应该能够通过呈现生化和物理线索来模拟内源性细胞外基质。新型水凝胶基材料寻求满足细胞相容性、可生物降解性、可打印性和在温和条件下交联的标准。然而，大多数现有的水凝胶缺乏细胞相互作用基序，这些基序对于调节细胞反应至关重要。将植物病毒 PVX 掺入水凝胶中可以改善整合素结合和矿化等功能，因为在粒子表面呈现肽。经过基因工程改造的 PVX 的定制表面形成原纤维样纳米结构，从而增强了依赖于焦点附着的细胞附着和基质矿化，并为开发用于骨组织替代物的新型水凝胶组成提供了巨大潜力。
